Systems and methods for measuring translation of target proteins in cells

ABSTRACT

The present invention relates to systems and methods for measuring the rate of translation of a target protein in cells, which are based on the detection of translation of one or more predetermined codon pairs during synthesis of the target protein. The detection is provided by a FRET signal emitted from labeled tRNA molecules which are juxtaposed during synthesis of the protein.

FIELD OF THE INVENTION

The present invention relates to systems and methods for measuring therate of translation of a target protein in cells, which are based on thedetection of translation of one or more predetermined codon pairs duringsynthesis of the target protein. The detection is provided by a FRETsignal emitted from labeled tRNA molecules which are juxtaposed duringsynthesis of the protein.

BACKGROUND OF THE INVENTION The Process of Protein Synthesis

Protein synthesis is one of the most central life processes. A proteinis formed by the linkage of multiple amino acids via peptide bonds,according to a sequence defined by the template messenger RNA (mRNA).Protein synthesis occurs in the ribosomes, the protein manufacturingplants of every organism and nearly every cell type.

Ribosomes are ribonucleoprotein particles consisting of a small andlarge subunit. In bacteria these subunits have sedimentationcoefficients of 30 and 50, and thus are referred to as “30S” and “50S”respectively; in eukaryotes the sedimentation coefficients are 40 and60. The translation system makes use of a large number of components,including inter alia the ribosome, initiation, elongation, terminationand recycling factors, transfer RNA, amino acids, aminoacyl synthetases,magnesium, and the product polypeptides.

Aminoacylated tRNAs read codons in ribosome-bound messenger RNAs andtransfer their attached amino acids to growing peptide chains. The tRNAmolecule has 73-93 nucleosides arranged in a cloverleaf-like structure,and includes the elements of an acceptor stem, a D-loop, an anticodonloop, a variable loop and a TψC loop. Aminoacylation, or charging, oftRNA results in linking the carboxyl terminal of an amino acid to the2′- (or 3′-) hydroxyl group of a terminal adenosine base via an esterlinkage. Aminoacylation occurs in two steps, amino acid activation (i.e.adenylation of the amino acid to produce aminoacyl-AMP), tRNAaminoacylation (i.e. attachment of an amino acid to the tRNA).

In most species, multiple tRNA molecules exist which becomeaminoacylated with the same amino acid but have different anticodonsequences. Such families of tRNAs are termed isoacceptors. There are 21isoacceptor families, corresponding to 20 for the standard amino acidsand one for seleno-cysteine. A particular tRNA may be denoted accordingto its aminoacylating amino acid, also referred to as its cognate aminoacid, which is indicated in superscript such as in tRNA^(Leu). Aparticular tRNA may further be denoted according to its anticodon,indicated in parentheses, such as tRNA^(Leu) (CAG).

According to the genetic code, the total possible number of anticodonsis 64, meaning that 61 different tRNA species (minus the stop codons)are theoretically required for translation. However, in most organismsthe total number of tRNA species is generally less than this. This isdue to the fact that at least some tRNAs are capable of reading (or“decoding”) different codons that encode the same amino acid. Forexample, E. coli has five tRNA^(Leu) isoacceptors that decode the sixleucine codons, and four tRNA^(Arg) isoacceptors that decode the sixarginine codons. Distinct codons that encode the same amino acid aretermed “synonymous” codons. Synonymous codons which differ only at thethird base may often be read by the same tRNA due to “wobble” pairing.Wobble pairing is base pairing between a codon and an anticodon thatdoes not obey the standard Watson-Crick pairing at the wobble position.

Protein translation, also referred to as “polypeptide synthesis,”involves the stages of initiation, elongation and termination. Theinitiation stage begins by formation of the initiation complex, composedof the two ribosomal subunits, protein initiation factors, mRNA, and aninitiator tRNA, which recognizes the initiator codon UAG of open readingframes. Elongation proceeds with repeated cycles of charged tRNAsbinding to the ribosome (a step termed “recognition”), peptide bondformation, and translocation, involving elongation factors and enzymessuch as peptidyl transferase, which catalyzes addition of amino acidmoieties onto the growing chain. Termination factors recognize a stopsignal, such as the base sequence UGA, in the mRNA, terminatingpolypeptide synthesis and releasing the polypeptide chain and mRNA fromthe ribosome. Recycling factor enables dissociation of the ribosomesubunits, which are then available for a new round of protein synthesis(see for example, Kapp et al., 2004, Annu Rev Biochem. 73:657-704). Ineukaryotes, ribosomes are often attached to the membranes of theendoplasmic reticulum (ER) and Golgi compartments. Additionally,ribosomes are active in organelles such as mitochondria and, in plantcells, in chloroplasts, and in other subcellular compartments. Oneimportant locus of protein synthesis activity is in dendritic spines ofneurons.

Stem Cell-Based Therapies for Congenital Diseases

Stem cells and stromal cells of various types are currently indevelopment for potential use in cell therapy for a wide variety ofcongenital diseases involving a deficiency of one or more proteinsinvolved in normal growth or metabolism. Such proteins include insulin,growth factors, and erythropoietin, among many others. Stem and stromalcell populations that secrete such proteins in therapeutic qualities areurgently sought after, as described for example in Soto-Gutierrez A etal, Acta Med Okayama. 2008 April; 62(2):63-8; and in Yokoo T et al,Transplantation. 2008 Jun. 15; 85(11):1654-8. Methods for monitoringproduction of the target protein from stem cell candidates are criticalto the development of such therapies.

In addition, stem and stromal cells have great potential in regenerativetherapy for a wide variety of disorders, as described in Caplan A I.,Adult mesenchymal stem cells for tissue engineering versus regenerativemedicine. J Cell Physiol. 2007 November; 213(2):341-7. In order tomonitor differentiation of stem and stromal cells into the desired celltypes, it is important to be able to measure production ofspecialization and differentiation markers and factors at the proteintranslation level.

Use of Cells for Antibody Production

Cultured cells are widely used for production of recombinant antibodiesfor various therapeutic uses. In order to optimize antibody production,it is important to be able to measure antibody production at the proteintranslation level.

Fast and efficient methods for measuring translation of a particularprotein of interest in real time are particularly useful in the aboveapplications relating to stem cells and antibody production.

Diseases Related to Protein Translation

Control of protein translation is implicated in a large number ofdiseases. For example, the family of central nervous system (CNS)disorders connected with protein synthesis disturbances in neuralspines. The family includes fragile X mental retardation, autism, agingand memory degeneration disorders such as Alzheimer's disease. Neuralspines and synapses contain their own protein synthesis machinery.Synaptic plasticity, underpinning the most basic neural functions ofmemory and learning, is dependent upon proper regulation of spinalprotein synthesis. Another important family of diseases directlyconnected to protein synthesis includes genetic disorders associatedwith the presence of premature termination codons (PTC) in the codingsequence of a critical protein, preventing its translation. Suchdiseases include Duchenne Muscular Dystrophy and a large family ofcongenital diseases. A small molecule known as PTC124 (Welch E M et al,Nature 2007 May 3; 447(7140):87-91) helps the ribosome slide over themutated codon, thereby producing the required protein, albeit at only at1-5% of normal concentrations. These amounts are often sufficient tosustain the life of an afflicted individual. PTC suppression has alsobeen achieved by introducing charged suppressor tRNA into a living cell,enabling readthrough suppression of the PTC-containing mRNA andaccumulation of the encoded protein (Sako et al, Nucleic Acids Symp Ser,50:239-240, 2006.

Multiple myeloma (MM) is a cancer of the plasma cells characterized byproliferation of malignant plasma cells and a subsequent overproductionof intact monoclonal immunoglobulin (IgG, IgA, IgD, or IgE) orBence-Jones protein (free monoclonal κ and λ light chains). Thisincurable disease accounted for a disproportionate 2% cancer-relateddeath rate in the United States in 2008 (Jemal A, et al. Cancerstatistics, 2008. CA Cancer J Clin. 2008; 58:71-96). While currenttreatments including autologous transplantation (Harousseau J L, et al.Best Pract Res Clin Haematol. 2005; 18:603-618) have resulted in animprovement in overall survival (Kumar S K, et al. Blood. 2008;111:2516-2520), there remains a need for delineating MM cell biology. Ithas been disclosed that overall proteasome activity of primary MM cellsinversely correlates with apoptotic sensitivity to proteasome inhibition(Bianchi G et al. Blood, 26 2009, Vol. 113, No. 13, 3040-3049). Theassociated mechanism apparently involves triggering of stress responseswhich are characterized by an increase in the clearance of misfoldedproteins by the 26S proteasome, an increase in the transcription andtranslation of chaperone proteins and foldases, and a strong decrease inglobal protein synthesis (Fribley A, et al. 2006. Cancer Biol Ther 5:745-748; Bush K T, et al. J Biol Chem 272 (14) 1997 9086-9092.

Hepatic fibrosis is a common response to most chronic liver injuries,including viral hepatitis, parasitic infection, metabolic and immunediseases, congenital abnormalities, and drug and alcohol abuse. It ischaracterized by increased production of fibril-forming collagen andassociated scar formation. Collagen is a family of proteins, wherein theconstituent chains are generally about 300 amino acids in length, andare highly enriched in Gly-Pro and Pro-Gly repeating units. Currently,collagen is measured with DNA microarrays, which indicate the existenceof collagen transcripts, but do not correlate well with actual synthesisof the protein.

In each of the above disease applications, it is important to rapidlyand efficiently measure in real-time translation of a one or moreparticular proteins of interest. In some cases, it is important to studythe subcellular localization of such proteins, as for example in thecase of local protein synthesis in neurons.

FRET Technology

Fluorescence resonance energy transfer (FRET) is a method widely used tomonitor biological interactions. FRET utilizes a donor fluorophore,having an emission spectrum that overlaps with the excitation spectrumof the acceptor fluorophore. Only when the donor fluorophore andacceptor fluorophore are in close proximity, typically about 10 nm, is asignal emitted from the acceptor fluorophore. FRET is described inSzollosi J, Damjanovich S, Mátyus L, Application of fluorescenceresonance energy transfer in the clinical laboratory: routine andresearch, Cytometry 34(4):159-79, 1998.

Existing Methods of Measuring Protein Translation

Cell assays that quantify production of a single specific proteintypically require cell lysis and/or permeabilization, making thenunsuitable for analysis of living cells. Further required are antibodiesor other specific reagents directed to the protein of interest inconjunction with various dyes and detection reagents. Further, suchmethods produce an estimation of the total production of proteins over agiven period of time measured in minute, hours or days. Those methodswhich are suitable for intact cells and organelles, such as metaboliclabeling with radioactive tracers, are unable to isolate a signal from aparticular transcript or protein of interest, and thus do not providereal-time or dynamic measurements of translation activity.

U.S. Pat. No. 6,210,941 to Rothschild discloses methods for thenon-radioactive labeling, detection, quantitation and isolation ofnascent proteins translated in a cellular or cell-free translationsystem. tRNA molecules are mis-aminoacylated with non-radioactivemarkers that may be non-native amino acids, amino acid analogs orderivatives, or substances recognized by the protein synthesizingmachinery. U.S. Patent Application Publication Nos. 2003/0219783 and2004/0023256 of Puglisi disclose compositions and methods for solidsurface translation, where translationally competent ribosome complexesare immobilized on a solid surface. According to the disclosure,ribosomes and one or more components of the ribosome complex may belabeled to permit analysis of single molecules for determination ofribosomal conformational changes and translation kinetics.

WO 2004/050825 of one of the inventors of the present inventiondiscloses methods for monitoring the synthesis of proteins by ribosomesin cells or a cell-free translation system, involving use of donor andacceptor fluorophores to label two locations of a ribosome, or each of aribosome and a tRNA, or each of a ribosome and an amino acid.

WO 2005/116252 of one of the inventors of the present inventiondiscloses methods for identifying ribonucleotide sequences of mRNAmolecules using immobilized ribosomes in a cell-free translation system.In the disclosed methods, a tRNA and a ribosome component are engineeredto carry donor and acceptor fluorophores or are used in FRET via theirnatural fluorescent properties.

The prior art does not disclose or suggest a method for measuring ratesof translation of a protein of interest in living cells which utilizes apair of tRNA molecules directly labeled with two components constitutinga FRET pair, wherein the pair of tRNAs corresponds to a specific pair ofadjacent amino acids occurring in the protein. There is an ongoing needfor such methods. Methods for measuring changes in protein synthesisrates in response to a drug candidate would be very useful for drugscreening and assays for predicting therapeutic activity of candidatedrugs. Also highly advantageous would be methods for measuringtranslation of a protein of interest that are amenable forhigh-throughput screening and cell sorting.

SUMMARY OF THE INVENTION

The present invention provides systems and methods for measuring proteintranslation and methods for real time measurements of translation of aselected protein of interest in viable cells and subcellularcompartments and organelles thereof.

The invention is based in part, on detection of an emitted FRET signalindicating translation of a predetermined codon pair within an mRNAsequence encoding a protein of interest. The FRET signal is produced bythe juxtaposition of two tRNAs, i.e. a tRNA pair, within the ribosomecomplex during protein translation. The two tRNAs selected to form thetRNA pair must correspond to tRNAs which read the adjacent codons on themRNA encoding the protein of interest. Each tRNA of the tRNA pair islabeled with a component of a FRET pair, namely either a FRET donorfluorophore or a FRET acceptor fluorophore.

As used herein, the term “tRNA pair” refers to a specific pair of tRNAspecies that are processed in consecutive order by a ribosome duringtranslation of an mRNA sequence.

Each tRNA member of the tRNA pair is capable of “reading” one codon ofthe codon pair. Thus, the cognate amino acid of each tRNA of the tRNApair corresponds to one amino acid encoded by a codon in the codon pair.

Without being bound by any theory or mechanism of action, the selectionof the tRNA pair is an important determinant for carrying out theinvention, since the signal emitted upon its translation must berepresentative of the mRNA encoding the specific protein of interest.Accordingly, in particular embodiments, the tRNA pair is preferably onewhich translates a codon pair which occurs at a relatively higherfrequency in the mRNA encoding the protein, as compared to theoccurrence of the same codon pair in other mRNAs being translated in thesame cell, tissue or organism under examination. Consequently, in somepreferred embodiments, the tRNA pair may be selected on the basis of theenrichment factor of the corresponding codon pair within the mRNAencoding the protein of interest.

The present invention advantageously provides readouts of the rate ofsynthesis of a selected protein of interest in a cell, including thatlocalized to a subcellular compartment of the cell. The presentinvention can be carried out in any type of cell, including primarycells and cell lines, with relatively mild intervention in the cellularmachinery and without requiring single ribosome analysis. The inventioncan be utilized for numerous applications, including, but not limitedto, diagnostic assays, macroscopic assays, microscopic assays,high-throughput screening, cell enrichment and cell sorting,applications in biomanufacturing, and in numerous proteinsynthesis-related diseases. Measurements of rates of protein translationcan be obtained in real time, which is crucial for instantly monitoringresponses to various stimuli, particularly changes in environmentalconditions, such as temperature, or exposure to specific compounds, suchas small molecule drug candidates, biotherapeutic agents, or any othersubstances which potentially affect protein synthesis. The presentinvention is thus advantageous for studying the effects of drugs anddrug candidates on cells in disease states characterized by aberrationsin protein translation, and for studying rates of synthesis of targettherapeutic proteins in stem cell and recombinant cell culture systems.

According to a first aspect, the present invention provides a system formeasuring translation of a protein of interest, the system comprising acell, wherein the cell comprises:

-   -   (ii) a nucleic acid sequence encoding a protein of interest,        wherein the nucleic acid sequence comprises at least one        predetermined codon pair; and    -   (ii) at least one tRNA pair, wherein for any one tRNA pair in        the system, each tRNA member of the tRNA pair is capable of        reading one codon of the at least one predetermined codon pair;        and further wherein one tRNA member of each tRNA pair is labeled        with a donor fluorophore and the other tRNA member of the tRNA        pair is labeled with an acceptor fluorophore, and wherein the        donor fluorophore and the acceptor fluorophore together form a        FRET pair.

In a particular embodiment, each tRNA member of a tRNA pair is a singletRNA species. In a particular embodiment, the cognate amino acid of eachtRNA member of a tRNA pair corresponds to one amino acid encoded by apredetermined codon pair. In a particular embodiment, the codon pairencodes a predetermined dipeptide occurring in the protein of interest.In a particular embodiment, the predetermined codon pair encodes aminoacids occurring in adjacent positions in the protein of interest. In aparticular embodiment, the amino acids occur in adjacent positions inthe protein of interest in the order from C-terminus to N-terminus, orin the order from N-terminus to C-terminus.

In a particular embodiment, the system comprises a single tRNA pair. Ina particular embodiment, the system comprises a plurality of tRNA pairs.In a particular embodiment, the plurality of tRNA pairs comprises thecomplete set of isoacceptor tRNAs specific for one amino acid encoded bya predetermined codon pair. In a particular embodiment, the plurality oftRNA pairs comprises a subset of isoacceptor tRNAs specific for oneamino acid encoded by a predetermined codon pair.

In a particular embodiment, the plurality of tRNA pairs comprise tRNAspecies that have cognate amino acids that are different one from theother.

In a particular embodiment, the plurality of tRNA pairs is selectedindependently of their cognate amino acids.

In a particular embodiment, the plurality of tRNA pairs comprises afirst complete set of isoacceptor tRNAs specific for a first amino acidencoded by a predetermined codon pair, and further comprises a secondcomplete set of isoacceptor tRNAs specific for a second amino acidencoded by the same predetermined codon pair. In a particularembodiment, each of the isoacceptor tRNAs of the first complete set arelabeled with the same donor fluorophore, and each of the isoacceptortRNAs of the second complete set are labeled with the same acceptorfluorophore. In a particular embodiment, each of the isoacceptor tRNAsof the first complete set are labeled with the same acceptorfluorophore, and each of the isoacceptor tRNAs of the second completeset are labeled with the same donor fluorophore.

In particular embodiments, the first complete set is labeled with aplurality of different donor fluorophores. In particular embodiments,the first complete set is labeled with a plurality of different acceptorfluorophores. In particular embodiments, the second complete set islabeled with a plurality of different donor fluorophores. In particularembodiments, the second complete set is labeled with a plurality ofdifferent acceptor fluorophores.

In a particular embodiment, the plurality of tRNA pairs comprises afirst subset of isoacceptor tRNAs specific for a first amino acidencoded by a predetermined codon pair, and further comprises a secondsubset of isoacceptor tRNAs specific for a second amino acid encoded bythe same predetermined codon pair. In a particular embodiment, each ofthe isoacceptor tRNAs of the first subset are labeled with the samedonor fluorophore, and each of the isoacceptor tRNAs of the secondsubset are labeled with the same acceptor fluorophore. In a particularembodiment, each of the isoacceptor tRNAs of the first subset arelabeled with the same acceptor fluorophore, and each of the isoacceptortRNAs of the second subset are labeled with the same donor fluorophore.

In a particular embodiment, the first subset is labeled with a pluralityof different donor fluorophores. In particular embodiments, the firstsubset is labeled with a plurality of different acceptor fluorophores.In particular embodiments, the second subset is labeled with a pluralityof different donor fluorophores. In particular embodiments, the secondsubset is labeled with a plurality of different acceptor fluorophores.

In a particular embodiment, the plurality of tRNA pairs comprises acomplete set of isoacceptor tRNAs specific for a first amino acidencoded by a predetermined codon pair, and further comprises a subset ofisoacceptor tRNAs specific for a second amino acid encoded by the samepredetermined codon pair. In a particular embodiment, each of theisoacceptor tRNAs of the complete set are labeled with the same donorfluorophore, and each of the isoacceptor tRNAs of the subset are labeledwith the same acceptor fluorophore. In a particular embodiment, each ofthe isoacceptor tRNAs of the complete set are labeled with the sameacceptor fluorophore, and each of the isoacceptor tRNAs of the subsetare labeled with the same donor fluorophore.

In a particular embodiment, the complete set is labeled with a pluralityof different donor fluorophores and the subset is labeled with aplurality of different acceptor fluorophores. In a particularembodiment, the complete set is labeled with a plurality of differentacceptor fluorophores and the subset is labeled with a plurality ofdifferent donor fluorophores.

Each possibility represents a separate embodiment of the presentinvention.

In a particular embodiment, the system comprises a plurality ofisoacceptor tRNAs specific for one amino acid encoded by a predeterminedcodon pair. In a particular embodiment, each of the isoacceptor tRNAsspecific for said one amino acid is labeled with the same donorfluorophore. In a particular embodiment, each of the isoacceptor tRNAsspecific for said one amino acid is labeled with the same acceptorfluorophore. In a particular embodiment, the plurality of isoacceptortRNAs specific for said one amino acid is labeled with a plurality ofdifferent acceptor fluorophores. In a particular embodiment, theplurality of isoacceptor tRNAs specific for said one amino acid islabeled with a plurality of different donor fluorophores.

Each possibility represents a separate embodiment of the presentinvention.

In a particular embodiment, the system comprises a first plurality ofisoacceptor tRNAs specific for a first amino acid that is encoded by onecodon of a predetermined codon pair, and further comprises a secondplurality of isoacceptor tRNAs specific for a second amino acid that isencoded by the other codon of the same predetermined codon pair. In aparticular embodiment, the first plurality is labeled with the samedonor fluorophore, and the second plurality is labeled with the sameacceptor fluorophore. In a particular embodiment, the first plurality islabeled with the same acceptor fluorophore, and the second plurality islabeled with the same donor fluorophore. In a particular embodiment, thefirst plurality of isoacceptor tRNAs is labeled with a plurality ofdifferent donor fluorophores and the second plurality of isoacceptortRNAs is labeled with a plurality of different acceptor fluorophores. Ina particular embodiment, the first plurality of isoacceptor tRNAs islabeled with a plurality of different acceptor fluorophores and thesecond plurality of isoacceptor tRNAs is labeled with a plurality ofdifferent donor fluorophores.

In a particular embodiment, one member of any one tRNA pair is capableof reading one codon of each of a plurality of predetermined codonpairs.

In a particular embodiment, one member of any one tRNA pair is capableof reading synonymous codons encoding a particular amino acid.

In particular embodiments, the cell is a live cell or a fixated cell. Inparticular embodiments, the cell is selected from the group consistingof human cells, non-human mammalian cells, vertebrate cells, aviancells, insect cells, yeast cells and plant cells.

In a particular embodiment, the system is for measuring translation of aprotein within a subcellular compartment of a live cell. In a particularembodiment, the tRNA pair labeled with the FRET pair is localized to asubcellular compartment. In particular embodiments, the subcellularcompartment is selected from the group consisting of dendritic spines,mitochondria, endoplasmic reticulum (ER) and chloroplasts.

In a particular embodiment, the nucleic acid sequence is an mRNAsequence.

In particular embodiments, the predetermined codon pair occurs at afrequency in the mRNA encoding the protein that is relatively higher, ascompared to the frequency of the translation product of the same codonpair in the proteome of the cell or a subcellular compartment thereof.In particular embodiments, the frequency of said translation product inthe proteome is that determined at a given time point.

In particular embodiments, the predetermined codon pair occurs at afrequency in the mRNA encoding the protein that is relatively higher, ascompared to the frequency of the same codon pair in the transcriptome ofthe cell or a subcellular compartment thereof.

In particular embodiments, the nucleic acid sequence encoding theprotein of interest has been altered to increase the frequency of apredetermined codon pair. In a particular embodiment, the nucleic acidsequence encoding the protein of interest has been altered to decreasethe frequency of a predetermined codon pair.

In particular embodiments, the predetermined codon pair occurs induplicate in adjacent positions in the mRNA encoding the protein ofinterest. In particular embodiments, opposite orientations of thepredetermined codon pair occur in adjacent positions in the mRNAencoding the protein of interest. In a particular embodiment, at leastone of the orientations of the predetermined codon pair occurs in themRNA encoding the protein of interest at a frequency that is relativelyhigher, as compared to the frequency of the same codon pair in thetranscriptome of the cell.

In a particular embodiment, the codons read by the tRNA pair togethercorrespond to an adjacent codon pair, wherein the adjacent codon pairoccurs within an mRNA transcript encoding the protein of interest at afrequency that is relatively higher, as compared to the frequency of thetranslation product of the same codon pair in the proteome of the cellor a subcellular compartment thereof. In a particular embodiment, thefrequency of said translation product in the proteome is that determinedat a given time point.

In a particular embodiment, the codons read by the tRNA pair togethercorrespond to an adjacent codon pair, wherein the adjacent codon pairoccurs within an mRNA transcript encoding the protein of interest at afrequency that is relatively higher, as compared to the same adjacentcodon pair in the transcriptome of the cell or a subcellular compartmentthereof.

In a particular embodiment, the system comprises two or more tRNA pairslabeled with two or more FRET pairs. In a particular embodiment, thesystem comprises two tRNA pairs, wherein each of the codon pairstranslated by the respective tRNA pairs occurs at a frequency in themRNA encoding the protein that is relatively higher, as compared to thefrequency of the translation product of the same codon pairs in theproteome of the cell. In a particular embodiment, the frequency of saidtranslation product in the proteome is that determined at a given timepoint.

In a particular embodiment, the system comprises two tRNA pairs, whereineach of the codon pairs translated by the respective tRNA pairs occursat a frequency in the mRNA encoding the protein that is relativelyhigher, as compared to the frequency of the same codon pairs in thetranscriptome of the cell.

In a particular embodiment, the system comprises two different tRNApairs, wherein each tRNA pair corresponds to a distinct codon pair, andwherein each distinct codon pair occurs in one of two different mRNAsrespectively encoding two different proteins of interest. In aparticular embodiment, the system comprises two different tRNA pairs andtwo different mRNAs respectively encoding two different proteins ofinterest, wherein each tRNA pair corresponds to a distinct codon pair inone of the two different mRNAs.

In a particular embodiment, each codon pair occurs in its respectivemRNA at a frequency that is relatively higher, as compared to theoccurrence of the translation product of the same codon pair in theproteome of the cell. In a particular embodiment, the frequency of saidtranslation product in the proteome is that determined at a given timepoint.

In a particular embodiment, one tRNA pair corresponds to a codon pairwhich occurs in its respective mRNA at a frequency that is relativelyhigher, as compared to the frequency of the translation product of thesame codon pair in the proteome of the cell. In a particular embodiment,the frequency of said translation product in the proteome is thatdetermined at a given time point.

In another embodiment, there is provided a system for sorting a cellpopulation based on the expression level of a protein of interest in thecells. The cell sorting system comprises the system of the inventiondescribed herein, a means of measuring a detectable signal produced in acell as a result of translation of the protein of interest, and a meansof sorting the cells.

Further provided is a system for enriching a cell population having aparticular level of expression of a protein of interest. The cellenriching system comprises the system of the invention described herein,a means of measuring a detectable signal produced in a cell as a resultof translation of the protein of interest, and a means of sorting thecells so as to enrich for the population of cells having the desiredexpression level of the protein.

In another aspect, the present invention provides a method for measuringtranslation of a protein of interest in a biological sample comprisingat least one cell, the method comprising the steps of:

-   -   (vi) selecting a first codon pair which occurs in the mRNA        sequence encoding a protein of interest at a known frequency;    -   (vii) introducing into the biological sample comprising cells at        least one first tRNA pair, wherein for each first tRNA pair,        each tRNA member of the pair is capable of reading one codon of        the first codon pair selected in (i); and wherein one tRNA        member of the tRNA pair is labeled with a donor fluorophore and        the other tRNA member of the tRNA pair is labeled with an        acceptor fluorophore, and wherein the donor fluorophore and the        acceptor fluorophore together form a first FRET pair; and    -   (viii) detecting FRET signals emitted from the biological sample        during protein translation, thereby measuring translation of the        protein of interest.

In particular embodiments, the codon pair selected in (i) occurs in themRNA sequence at a frequency that is relatively higher, as compared tothe frequency of the translation product of the same codon pair in theproteome of the cell, or a subcellular compartment thereof. Inparticular embodiments, the frequency of said translation product isthat determined at a given time point.

Additional particular embodiments of the cell, the codon pair, the tRNApair and the FRET pair are as hereinbefore described.

In a particular embodiment, the biological sample is selected from thegroup consisting of a cell line, a primary cell culture and a wholeorganism. In a particular embodiment, the step of introducing is intocells selected from the group consisting of human cells, non-humanmammalian cells, vertebrate cells, avian cells, insect cells, yeastcells and plant cells. In a particular embodiment, the biological samplecomprises diseased cells. In a particular embodiment, the step ofintroducing is into a subcellular compartment of living cells. Inparticular embodiments, the subcellular compartment is selected from thegroup consisting of dendritic spines, mitochondria, endoplasmicreticulum (ER) and chloroplasts.

In a particular embodiment, the whole organism is a model organism usedfor genetic research. In another embodiment, the model organism isselected from the group consisting of Escherichia. coli, Caenorhabditiselegans, Drosophila melanogaster, Arabidopsis thaliana, and an inbredstrain of Mus musculus.

In a particular embodiment, the method further comprises a step ofirradiating the biological sample with electromagnetic radiation priorto the step of detecting the emitted FRET signals, wherein theelectromagnetic radiation used for irradiating is of a wavelengthdifferent from the detected FRET signals.

In a particular embodiment, the method further comprises a step ofaltering the nucleic acid sequence of the gene encoding the protein ofinterest so as to increase the frequency of the codon pair in the mRNAsequence encoding the protein.

In a particular embodiment, the method further comprises:

-   -   (ix) selecting a second codon pair, wherein the second codon        pair occurs in the mRNA sequence encoding the protein of        interest at a frequency that is relatively higher, as compared        to the frequency of the translation product of the second codon        pair in the proteome of the cell; and    -   (x) introducing into the biological sample at least one second        tRNA pair, wherein each tRNA member of the pair is capable of        reading one codon of the second codon pair selected in (iv),        wherein each tRNA member of the second tRNA pair is labeled with        either a second donor fluorophore or a second acceptor        fluorophore, and wherein the second donor fluorophore and the        second acceptor fluorophore together form a second FRET pair.

In a particular embodiment of the method, each of the at least one firsttRNA pair and the at least one second tRNA pair comprises a distinctplurality of tRNA pairs. The plurality of tRNA pairs relating to thefirst tRNA pair is referred to herein as “the first tRNA pairplurality”; and the plurality of tRNA pairs relating to the second tRNApair is referred to herein as “the second tRNA pair plurality”.

In a particular embodiment, the first tRNA pair plurality comprises thecomplete set of isoacceptor tRNAs specific for one amino acid encoded bythe first codon pair or a subset of said isoacceptor tRNAs. Eachpossibility represents a different embodiment of the invention. In aparticular embodiment, the first tRNA pair plurality comprises acomplete set of isoacceptor tRNAs specific for each of the two aminoacids encoded by the first codon pair.

In a particular embodiment, the second tRNA pair plurality comprises thecomplete set of isoacceptor tRNAs specific for one amino acid encoded bythe second codon pair or a subset of said isoacceptor tRNAs. Eachpossibility represents a different embodiment of the invention. In aparticular embodiment, the second tRNA pair plurality comprises acomplete set of isoacceptor tRNAs specific for each of the two aminoacids encoded by the second codon pair.

In a particular embodiment, each distinct plurality of tRNA pairscomprises tRNA species that have cognate amino acids that are differentone from the other. In a particular embodiment, each of the isoacceptortRNAs of the first tRNA pair plurality are labeled with the same donorfluorophore or with a plurality of different donor fluorophores. In aparticular embodiment, each of the isoacceptor tRNAs of the second tRNApair plurality are labeled with the same acceptor fluorophore or with aplurality of different acceptor fluorophores. In a particularembodiment, each of the isoacceptor tRNAs of the first tRNA pairplurality are labeled with the same acceptor fluorophore or with aplurality of different acceptor fluorophores. In a particularembodiment, each of the isoacceptor tRNAs of the second tRNA pairplurality are labeled with the same donor fluorophore or with aplurality of different donor fluorophores. Each possibility is adifferent embodiment of the invention.

In a particular embodiment, the system comprises distinct subsets ofisoacceptor tRNAs specific for each amino acid encoded by the firstcodon pair and the second codon pair.

In a particular embodiment, each distinct subset of isoacceptor tRNAs islabeled with a single donor fluorophore or with a plurality of differentdonor fluorophores. In a particular embodiment, each distinct subset ofisoacceptor tRNAs is labeled with a single acceptor fluorophore or witha plurality of different acceptor fluorophores. Each possibility is adifferent embodiment of the invention.

In a particular embodiment, the introducing of the second tRNA pair in(v) is into a cell or subcellular compartment that is other than thatinto which the first tRNA pair is introduced in (ii).

In a particular embodiment, the first codon pair occurs in the mRNAsequence of the protein of interest at a frequency that is relativelyhigher, as compared to the frequency of the translation product of thesame codon pair in the proteome of a subcellular compartment of thecell, and the introducing of the first tRNA pair in (ii) is into thesame subcellular compartment

In a particular embodiment, at least one of the acceptor fluorophore andthe donor fluorophore of the second FRET pair is other than that of thefirst FRET pair. In a particular embodiment, the acceptor fluorophoreand the donor fluorophore of the second FRET pair are both other thanthat of the first FRET pair.

In a particular embodiment, the method further comprises altering anucleic acid sequence of the gene encoding the protein so as to increasethe frequency of the second codon pair in the mRNA sequence encoding theprotein of interest.

In a particular embodiment, opposite orientations of the second codonpair occur in adjacent positions in the mRNA sequence encoding theprotein of interest.

In one embodiment, the selecting in (i) is of a codon pair which occursin the mRNA sequence of the protein of interest at a frequency that isrelatively higher, as compared to the frequency of the translation ofthe same codon pair in the proteome of the cell, and the introducing ofthe tRNA pair in (ii) is into the cell.

In a particular embodiment, the subcellular compartment is an organelle.In another embodiment, the detected FRET signals are quantitated,thereby providing a readout in real-time of the amount of translation ofthe protein of interest.

In particular embodiments, the methods of the invention further comprisea step of analyzing the detected FRET signals, thereby obtaining aread-out of translation of the protein of interest. In particularembodiments, analysis of the signals produces a read-out of a parameterof translation activity of the protein of interest.

In particular embodiments, the methods further comprise a step ofanalyzing the detected FRET signals, thereby obtaining an estimate ofthe translation of the protein of interest. In particular embodiments,the step of analyzing comprises the step of computing the number ofevents (N) over a period of time t, wherein

$\left. N \right.\sim\frac{\Sigma \; I_{t}^{2}}{\Sigma \; \delta \; I_{t}^{2}}$

wherein I_(t) is the average signal strength at time t and δI_(t) is theaverage signal deviation at time t. Each possibility represents aseparate embodiment of the present invention.

In particular embodiments, there is provided a method for sorting a cellpopulation based on the expression level of a protein, the methodcomprising performing a method described herein in intact cells andfurther comprising a step of sorting said cells so as to obtain a sortedcell population.

In particular embodiments, there is provided a method for enriching fora cell population based on the expression level of a protein, the methodcomprising performing a method described herein in intact cells andfurther comprising a step of sorting said cells so as to obtain a cellpopulation enriched in the expression level of the protein.

According to some embodiments, the methods of the present inventionfurther comprise a step of comparing the amount of detected FRET signalsto a reference standard. In another embodiment, a level of detected FRETsignals different from the reference standard is indicative of a diseaseor disorder. In another embodiment, the level of detected FRET signalsis diagnostic for a disease, disorder or pathological condition. Inanother embodiment, the readout provided upon analysis of said detectedFRET signals is diagnostic for a disease, disorder or pathologicalcondition. Thus, methods of the present invention can be used to detectin a subject a condition selected from the group consisting of adisease, a disorder and a pathological condition.

According to one embodiment, the condition is selected from the groupconsisting of fragile X mental retardation, autism, aging and memorydegeneration.

According to another embodiment, the disease is selected from the groupconsisting of a mitochondria-related disease, cardiac hypertrophy,restenosis, diabetes, obesity, a genetic disease resulting from apremature termination codon (PTC), and inflammatory bowel disease.

According to another embodiment, the pathological condition is amalignant or pre-malignant condition. In one embodiment, the malignantor pre-malignant condition is in a hematological cell. In oneembodiment, the malignant condition is a hematological malignancy. Inone embodiment, the hematological malignancy is selected from the groupconsisting of acute lymphoblastic leukemia (ALL); acute myelogenousleukemia (AML); chronic myelogenous leukemia (CML); Hodgkin's disease;non-Hodgkin lymphoma; chronic lymphocytic leukemia (CLL); diffuse largeB-cell lymphoma (DLBCL); follicular lymphoma (FL); mantle cell lymphoma(MCL); hairy cell leukemia (HCL); marginal zone lymphoma (MZL);Burkitt's lymphoma (BL); post-transplant lymphoproliferative disorder(PTLD); T-cell prolymphocytic leukemia (T-PLL); B-cell prolymphocyticleukemia (B-PLL); Waldenstrom's macroglobulinemia and multiple myeloma(MM). In one embodiment, the malignant disorder is multiple myeloma.

In one embodiment, the pre-malignant condition is selected from thegroup consisting of monoclonal gammopathy of uncertain significance andsmoldering multiple myeloma.

In other embodiments, a method of the present invention furthercomprises the step of administering to a cell or subcellular compartmentat least one drug candidate prior to the step of detecting emitted FRETsignals. According particular embodiments, the method is performed onseparate biological samples, wherein the samples are substantiallyidentical, except that one sample is analyzed following contact with thedrug candidate, and the other sample has not been contacted with thedrug candidate. “Substantially identical” as used herein refers to theabsence of apparent differences between the biological samples. Anon-limiting example of biological samples that are substantiallyidentical is a set of two different aliquots from the same preparationof cells or subcellular organelles. Each possibility represents aseparate embodiment of the present invention.

In other embodiments, the above method further comprises the step ofcomparing the intensities of FRET signals emitted from the twobiological samples, i.e. those contacted and not contacted with the drugcandidate. In other embodiments of this method, a difference betweenthese two intensities, in particular a statistically significantdifference, indicates that the drug candidate affects proteintranslation. Each possibility represents a separate embodiment of thepresent invention.

According to other embodiments, a method of the present inventionfurther comprises the steps of (a) administering to the biologicalsample a drug candidate; (b) detecting the FRET signals emitted by thebiological sample, as described herein; and (c) comparing the FRETsignals detected in the presence of the drug candidate, to that detectedin the absence of the drug candidate, thereby evaluating the effect ofthe drug candidate on protein translation.

According to a particular embodiment, a method for measuring the effectof a drug candidate on translation of a protein of interest comprisesthe steps of:

-   -   (vi) introducing into a first biological sample a tRNA pair        labeled with a FRET pair, wherein an acceptor fluorophore and a        donor fluorophore together form the FRET pair and wherein each        member of the tRNA pair is labeled with one of the acceptor        fluorophore or the donor fluorophore, and wherein each member of        the tRNA pair is capable of reading one codon of a predetermined        codon pair, wherein the biological sample further comprises an        mRNA transcript encoding the protein of interest, and wherein        the predetermined codon pair occurs in the mRNA transcript;    -   (vii) providing a second biological sample that is substantially        identical to the first biological sample;    -   (viii) introducing a drug candidate to one of the first        biological sample and the second biological sample; and    -   (iv) measuring FRET signals emitted from each of the first and        second biological samples, wherein a significant difference        between the FRET signals measured in the first and second        biological samples indicates that the drug candidate affects the        translation of the protein of interest.

In other embodiments, methods and systems of the present invention areused for high-throughput-screening (HTS) of putative translationmodulators.

According to a particular embodiment, the codon pair appears in the mRNAsequence encoding the protein of interest at a frequency that is higher,as compared to the frequency of—the translation product of the samecodon pair in the proteome of the cell or a subcellular compartmentthereof. According to a particular embodiment, the codon pair occurs inthe mRNA sequence encoding the protein of interest at a frequency thatis higher, as compared to the frequency of the same codon pair in thetranscriptome of the cell or a subcellular compartment thereof.

According to particular embodiments, the drug candidate of the presentinvention is selected from the group consisting of a small molecule, apeptide, an enzyme, a hormone, and a biotherapeutic agent.

According to another aspect, there is provided a method for measuringthe effect of a drug candidate on translation of a protein of interestin a biological sample, said method comprising the steps of:

-   -   (ix) selecting a specific codon pair which occurs in the mRNA        sequence encoding a protein of interest at a frequency that is        relatively higher, as compared to the frequency—of the        translation product of the same codon pair in the proteome of        the biological sample;    -   (x) introducing into the biological sample a tRNA pair labeled        with a FRET pair, wherein an acceptor fluorophore and a donor        fluorophore together form the FRET pair and wherein each member        of the tRNA pair is labeled with one of the acceptor fluorophore        or the donor fluorophore, and wherein each member of the tRNA        pair is capable of reading one codon of a codon pair appearing        in the mRNA sequence encoding the protein of interest, and        wherein the biological sample further comprises a nucleic acid        sequence encoding said protein of interest;    -   (xi) measuring FRET signals emitted from the biological sample;    -   (xii) contacting the biological sample with a drug candidate;        and    -   (xiii) measuring FRET signals emitted from said acceptor        fluorophore, under the conditions of (iv), wherein a difference        between the FRET signals measured in step (iii) and the FRET        signals measured in step (v) indicates that said drug candidate        affects translation of the protein of interest.

In a particular embodiment, the biological sample is selected from thegroup consisting of a cell and a subcellular compartment thereof.

According to other embodiments of the methods and systems of the presentinvention, the protein of interest is selected from the group consistingof insulin, a growth factor, an antibody, erythropoietin, and a stemcell specialization factor.

According to some embodiments, the protein of interest further comprisesa protein tag. According to other embodiments, the protein of interestis a fusion protein comprising a protein tag. According to otherembodiments, the protein tag is selected from the group consisting of anaffinity tag, a solubilization tag, a chromatography tag, an epitope tagand a fluorescent protein tag. According to some embodiments, thetranslation product of the tRNA pair occurs in the protein tag.

These and other embodiments of the present invention will becomeapparent in conjunction with the figures, description and claims thatfollow.

BRIEF DESCRIPTION OF THE FIGURES

The invention is herein described, by way of example only, withreference to the accompanying figures. With specific reference now tothe figures in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe figures making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

FIG. 1 is a schematic presentation of a bacterial ribosome structurewith the large (50S) subunit 20, small (30S) subunit 25, aminoacyl (A)site 50 where the tRNAs are initially docked, peptidyl (P) site 51 wherethe growing polypeptide chain is docked, and exit (E) site 52 wherefromthe deacylated tRNA is removed once the cycle is complete. On the rightside, tRNAs that are still undocked, i.e. 40, 41, 42 and 43, aredepicted. mRNA being decoded 30 and the nascent polypeptide chain beingsynthesized 45 are also depicted. The ribosome itself is made up oflarge folded rRNA chains with ribosomal proteins. The large subunit 20contains two folded rRNAs, known as 23S and 5S. The small subunit 25contains one folded rRNA, 30S (not shown). On the folded rRNA chainsmore than 50 ribosomal proteins are docked (not shown). They arecustomarily denoted by L1, L2 etc. for the approximately 36 ribosomalproteins attached to the large subunit, and by S1, S2 etc. for theapproximately 21 ribosomal proteins attached to the small subunit(numbers given are for E. coli ribosomes).

FIG. 2 is an exemplary overview of one preferred embodiment for signalgeneration and analysis. Illumination module 350 illuminates sample 354through microscope 352, and the resulting signals are detected bydetection module 356. The resultant image can then be transferred tocomputerized analysis station 360 which analyzes the images, preferablyrecords the produced signals, and analyzes them to produce an estimationof the specific measurement that is required. The readout can bepresented on the computer screen and if desired stored in database 362for further analysis.

FIG. 3 is an illustration of a modified standard electrophysiology setupused for electroporation-mediated transfection. The culture is placed ina perfusion chamber and visualized using gradient-contrast illuminationand IR video microscopy with a 40× water immersion objective and further2× magnification. Individual neurons can be identified on the monitorscreen. The DNA filled micropipette can be targeted precisely to themembrane of a single soma. A back-pressure of 6-7 mbar is applied to thepipette. Two hundred 1 ms-long square pulses with an interpulse delay of4 ms and an amplitude of 10 V are delivered to each neuron.

FIG. 4 is a schematic presentation of tRNA-tRNA FRET. An exemplarymethod of attaching a fluorophore to tRNA is depicted. Stars denotefluorescent labels on the D-loop of each tRNA. Arrows (from right toleft) denote excitation of the donor, energy transfer to acceptor(dotted gray arrow), and emission from the acceptor, respectively.

FIG. 5A shows the results of ensemble tRNA:tRNA FRET. Fluorescencespectra of PRE complexes formed with the donor-acceptor (DA)[Phe-tRNA^(Phe) (Cy3)+fMet-tRNA^(fmet) (Cy5)]; the donor alone (DU)[Phe-tRNA^(Phe) (Cy3)+unlabeled fMet-tRNA^(fmet)], and the acceptoralone (UA) [Phe-tRNA^(Phe)+fMet-tRNA^(fmet) (Cy5)] samples. The upperand middle traces are fluorescence intensities monitored at acceptor(680±10 nm) and donor (570±10 nm) wavelengths, respectively. The lowertrace measures the change in acceptor fluorescence in the presence ofadded viomycin.

FIG. 5B shows the results of ensemble tRNA:tRNA FRET. Fluorescencespectra of POST complexes formed with the donor-acceptor (DA)[Phe-tRNA^(Phe) (Cy3)+fMet-tRNA^(fmet) (Cy5)]; the donor alone (DU)[Phe-tRNA^(Phe) (Cy3)+unlabeled fMet-tRNA^(fmet)], and the acceptoralone (UA) [Phe-tRNA^(Phe)+fMet-tRNA^(fmet) (Cy5)] samples. The upperand middle traces are fluorescence intensities monitored at acceptor(680±10 nm) and donor (570±10 nm) wavelengths, respectively. The lowertrace measures the change in acceptor fluorescence in the presence ofadded viomycin.

FIG. 6A shows events excised from traces of real-time single moleculefluorescence traces of a single ribosome translating an mRNA of sequenceMRFVRFVRF (SEQ ID NO:10). For the experiment, tRNA^(Arg) was labeledwith Cy3 (donor) and tRNA^(Phe) was labeled with Cy5 (acceptor). Thetraces show the donor emission under donor excitation (legend: donor),acceptor emission under acceptor excitation (legend: acceptor) and FRET,or sensitized emission, which is acceptor emission under donorexcitation (legend: FRET).

FIG. 6B shows events excised from traces of a ribosome translating anmRNA of sequence MFRVFRVFR (SEQ ID NO:11). The figures show eventsexcised from traces of single ribosome experiments. For the experiment,tRNA^(Avg) was labeled with Cy3 (donor) and tRNA^(Phe) was labeled withCy5 (acceptor). The traces show the donor emission under donorexcitation (legend: donor), acceptor emission under acceptor excitation(legend: acceptor) and FRET, or sensitized emission, which is acceptoremission under donor excitation (legend: FRET).

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the term “tRNA” refers to transfer ribonucleic acid.Specific tRNAs recognize specific codons in mRNA, generally involvingstandard base pairing between the mRNA codon and the tRNA anticodon, andin some cases involving wobble pairing between codon and anticodon. An“initiator tRNA” is a specific tRNA molecule that is used only for theinitial amino acid of a synthesized polypeptide. A “suppressor tRNA” isa tRNA molecule that comprises an anticodon which allows pairing with atermination codon (e.g. UAG and UAA). An “elongator tRNA” is a tRNAmolecule that is neither an initiator nor a suppressor, and that placesits corresponding amino acid (i.e. cognate amino acid) in its propersequence during the process of translation.

The terms “cognate amino acid” and “aminoacylating amino acid” are usedherein interchangeably to refer to the specific amino acid that iscarried by a particular elongator tRNA.

A tRNA is referred to as “charged” or “aminoacylated” when it iscovalently associated with its cognate amino acid at the 3′ terminal CCAend.

In most species, multiple tRNA molecules exist which are capable ofbeing aminoacylated with the same amino acid, but have differentanticodon sequences. Such families of tRNAs are termed “isoacceptorfamilies”. There are 21 isoacceptor families, corresponding to 20 forthe standard amino acids and one for seleno-cysteine. In humans, 49specific tRNA molecules exist (including that carrying seleno-cysteine),wherein 1 to 5 different tRNA molecules may be charged with a specificamino acid.

As used herein, the terms “isoaccepting tRNAs” and “isoacceptors” mayencompass either the entire set of isoacceptors in an isoacceptorfamily, or a partial set i.e. subset, of isoacceptors in an isoacceptorfamily.

Reference herein to a tRNA specific for a particular amino acid mayencompass all of the isoaccepting tRNAs for that amino acid, a subset ofsuch isoacceptors, or a single tRNA capable of being aminoacylated bythat amino acid, unless the context expressly specifies a single tRNA.

A particular tRNA may be denoted herein according to its aminoacylatingamino acid, which is indicated in superscript such as in tRNA^(Leu). Aparticular tRNA may additionally be denoted according to its anticodon,indicated in parentheses, such as tRNA^(Leu) (CAG).

A particular tRNA may further be denoted herein in abbreviated form asXi, wherein X is the single letter identification of its cognate aminoacid and i is a numerical identifier (1≦i≦5) pertaining to the anticodonof the specific tRNA molecule and the codon(s) read by that tRNAmolecule. Table 1 herein lists Xi identifiers for tRNAs of Homo sapiens.

TABLE 1 Characteristics of the tRNAs occurring in_Homo sapiens (excluding that specific for seleno-cysteine) tRNA anti-codon1 codon2 (wobble) No. identifier codon read read 49. A₁ AGC GCT GCC50. A₂ CGC GCG 51. A₃ TGC GCA 52. G₁ GCC GGC GGT 53. G₂ CCC GGG 54. G₃TCC GGA 55. P₁ AGG CCT CCC 56. P₂ CGG CCG 57. P₃ TGG CCA 58. T₁ AGT ACTACC 59. T₂ CGT ACG 60. T₃ TGT ACA 61. V₁ AAC GTT GTC 62. V₂ CAC GTG 63.V₃ TAC GTA 64. S₁ AGA TCT TCC 65. S₂ CGA TCG 66. S₃ TGA TCA 67. S₄ GCTAGC AGT 68. R₁ ACG CGT CGC 69. R₂ CCG CGG 70. R₃ TCG CGA 71. R₄ CCT AGG72. R₅ TCT AGA 73. L₁ AAG CTT CTC 74. L₂ CAG CTG 75. L₃ TAG CTA 76. L₄CAA TTG 77. L₅ TAA TTA 78. F₁ GAA TTC TTT 79. N₁ ATT AAT 80. N₂ GTT AATAAC 81. K₁ CTT AAG 82. K₂ TTT AAA 83. D₁ GTC GAC GAT 84. E_(l) CTC GAG85. E₂ TTC GAA 86. H₁ GTG CAC CAT 87. Q₁ CTG CAG 88. Q₂ TTG CAA 89. I₁AAT ATT 90. I₂ GAT ATC 91. I₃ TAT ATA 92. M₁ CAT ATG 93. Y₁ ATA TAT 94.Y₂ GTA TAC TAT 95. C₁ GCA TGC TGT 96. W₁ CCA TGG

The terms “reading” and “decoding” are used herein interchangeably torefer to the process in which a tRNA recognizes a particular codon on anmRNA during its translation on a ribosomal complex.

The term “synonymous codons” is used herein to refer to distinct codonsthat encode the same amino acid. Synonymous codons which differ only atthe third base may often be read by the same tRNA due to “wobble”pairing i.e., base pairing between a codon and an anticodon that doesnot obey the standard Watson-Crick pairing at the wobble position.

The term “tRNA pair” as used herein refers to a specific pair of tRNAsthat are processed in consecutive order by a ribosome, as it synthesizesa protein by translating an mRNA molecule. Thus, for example, the twotRNA members of a tRNA pair will be held at the A and P sites of theribosomal complex at a substantially close or identical time pointduring the process of translation.

The term “tRNA sequence” as used herein refers to a sequence of tRNAsused for translation of a particular mRNA molecule during synthesis of aparticular protein, wherein the members of the sequence are processed ina consecutive manner by a ribosomal complex. A tRNA sequence may bedenoted herein by a sequence of Xi identifiers, for example thoseindicated in Table 1, according to the order of the tRNAs used for theprocessing of the particular mRNA.

The terms “adjacent codon pair” and “codon pair” are used hereininterchangeably to two distinct codons that appear in consecutive orderin a nucleic acid sequence, such as an mRNA, either in the directionfrom 5′ to 3′, or in the direction from 3′ to 5′.

The term “frequency” in reference to a codon or codon pair, refers tothe number of occurrences of the codon or codon pair within a particularnucleic acid sequence, such as a specific mRNA sequence, or within a setof nucleic acid sequences, expressed as a percentage of the total numberof codons or codon pairs occurring in the same nucleic acid sequence orset of nucleic acid sequences.

The term “enriched” in reference to a codon pair, means that theadjacent occurrence of the codons constituting the pair within aparticular nucleic acid sequence, such as a specific mRNA sequence, isat higher frequency compared to the occurrence of the same codon pair ina reference nucleic acid sequence or set of nucleic acid sequences.

The term “enriched” in reference to a tRNA pair, means that the tRNApair appears at higher frequency in the tRNA sequence of a particularprotein, as compared to the occurrence of the same tRNA pair in areference tRNA sequence or a set of tRNA sequences.

As used herein, the term “FRET” (“fluorescence resonance energytransfer”; also known as “Förster resonance energy transfer”) refers toa physical phenomenon involving a donor fluorophore and a matchingacceptor fluorophore selected so that the emission spectrum of the donoroverlaps the excitation spectrum of the acceptor, and further selectedso that when donor and acceptor are in close proximity (usually lessthan 10 nm), excitation of the donor will cause excitation of andemission from the acceptor, as some of the energy passes from donor toacceptor via a quantum coupling effect. Thus, a FRET signal serves as aproximity gauge of the donor and acceptor; only when they are in closeproximity is a signal generated. The donor fluorophore and acceptorfluorophore are collectively referred to herein as a “FRET pair”.

The terms “mRNA”, “transcript” and “mRNA transcript” are usedinterchangeably herein to describe a ribonucleotide sequence thattransfers genetic information to ribosomes, where it serves as atemplate for peptide or protein synthesis. Ribonucleotide sequences arepolymers of ribonucleic acids, and are constituents of all living cellsand many viruses. They consist of a long, usually single-stranded chainof alternating phosphate and ribose units with the bases adenine,guanine, cytosine, and uracil bonded to the ribose. The structure andbase sequence of RNA are determinants of protein synthesis and thetransmission of genetic information. In general, a “transcript” refersto a specific mRNA encoding a peptide or protein of interest.

As used herein, the terms “codon” and “triplet codon” referinterchangeably to the tri-nucleotide unit of a nucleic acid, such asmRNA, that encodes i.e. specifies a single amino acid. A nucleic acidsequence that encodes a specific peptide or protein is composed of astring of codons specifying the amino acid sequence of the protein.There are 4³=64 different codon combinations possible with a tripletcodon of three nucleotides; all 64 codons are assigned for either aminoacids or stop signals during translation.

As used herein, the terms “protein” and “polypeptide” referinterchangeably to a complex polymer compound composed of one or morelinear chains of amino acids, wherein the amino acids are covalentlyjoined together by peptide bonds between the carboxyl and amino groupsof adjacent amino acid residues. The sequence of amino acids in aprotein is determined by the corresponding nucleic acid sequence of thegene encoding the protein. Proteins include glycoproteins, antibodies,non-enzyme proteins, enzymes, hormones and peptides.

As used herein, the term “peptide” refers to a small to intermediatemolecular weight chain of amino acids covalently joined by peptide bondsbetween adjacent amino acid residues. Peptides generally have from 2 to100 amino acid residues and frequently but not necessarily represent afragment of a larger protein. Peptides may be the products of nativegenes, produced either naturally, by recombinant techniques or chemicalsynthesis. Alternately, peptides may be non-naturally occurringsequences.

As used herein, “one member of the tRNA pair” refers to an individualtRNA species of a tRNA pair. The members of the tRNA pair may be thesame or different tRNA species, but will be differentially labeled withacceptor fluorophore or donor fluorophore of a FRET pair.

As used herein, “cell” refers to a prokaryotic or a eukaryotic cell.Suitable cells can be, for example, of mammalian, avian, insect,bacterial, yeast or plant origin. Non-limiting examples of mammaliancells include human, bovine, ovine, porcine, murine, and rabbit cells.In another embodiment, the cell can be an embryonic cell, bone marrowstem cell, or other progenitor cell. In another embodiment, the cell isa somatic cell, which can be, for example, an epithelial cell,fibroblast, smooth muscle cell, blood cell (including a hematopoieticcell, red blood cell, T-cell, B-cell, etc.), tumor cell, cardiac musclecell, macrophage, dendritic cell, neuronal cell (e.g., a glial cell orastrocyte), or pathogen-infected cell (e.g., those infected by bacteria,viruses, virusoids, parasites, or prions).

The term “test cell” as used herein, refers to cells that aremanipulated for use in the translation assay of the invention.

The term “host cell” as used herein refers to cells that do notnaturally contain the labeled elements of the invention.

As used herein, the term “biological sample” refers to any of: anorganism, an organ, a tissue, a cell, a subcellular compartment or anorganelle; or a portion, fragment or aliquot thereof, which is used inthe methods and assays of the invention.

As used herein, the term “subcellular compartment” refers to any definedpart of the cell where protein translation activity takes place, such asdendritic spines, mitochondria, endoplasmic reticulum (ER) andchloroplasts.

As used herein, the term “organelle” refers to cellularmembrane-encapsulated structures such as the chloroplast, endoplasmicreticulum (ER) and mitochondrion.

As used herein, “introducing” refers to the transfer of molecules suchas ribosomes, tRNAs, translation factors and amino acids from outside ahost cell or subcellular compartment to inside a host cell orsubcellular compartment. Said molecules can be “introduced” into a hostcell or subcellular compartment by any means known to those of skill inthe art, for example as taught by Sambrook et al. Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, New York (2001).Means of “introducing” molecules into a host cell or subcellularcompartment include, but are not limited to heat shock, calciumphosphate transfection, electroporation, lipofection, and viral-mediatedtransfer.

The terms “polynucleotide”, “nucleic acid” and “nucleic acid sequence”refer interchangeably to a polymeric form of nucleotides at least 10bases in length. A polynucleotide may be present either in its genomicform or as an isolated polynucleotide. By “isolated polynucleotide” ismeant a polynucleotide that is not immediately contiguous with both ofthe coding sequences with which it is immediately contiguous (one on the5′ end and one on the 3′ end) in the naturally occurring genome of theorganism from which it is derived. The term therefore includes, forexample, a recombinant DNA which is incorporated into a vector; into anautonomously replicating plasmid or virus; or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (eg., acDNA) independent of other sequences. The nucleotides of the inventioncan be ribonucleotides, deoxyribonucleotides, or modified forms ofeither nucleotide. The term includes single and double stranded forms ofDNA.

As used herein, the term “transfection” refers to introduction of anucleic acid sequence into the interior of a membrane-enclosed space ofa living cell, including introduction of the nucleic acid sequence intothe cytosol of a cell as well as the interior space of a mitochondria,endoplasmic reticulum (ER) or chloroplast. The nucleic acid may be inthe form of naked DNA, RNA, or tRNA. The DNA, RNA, or tRNA is in someembodiments associated with one or more proteins. In another embodiment,the nucleic acid is incorporated into a vector. Each possibilityrepresents a separate embodiment of the present invention.

As used herein, the term “infection” means the introduction of a nucleicacid such as DNA, RNA, tRNA into a recipient cell, subcellularcompartment, or organism, by means of a virus. Viral infection of a hostcell is a technique that is well established in the art and is describedin a number of laboratory texts and manuals such as Sambrook et al.,Molecular Cloning: A Laboratory Manual, Vol. 1-3, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 2001.

As used herein, the terms “label”, “detectable label” and “tag” referinterchangeably to a molecule that is attached to or associated withanother molecule and that can be directly (i.e., a primary label) orindirectly (i.e., a secondary label) detected. For example, a label canbe visualized and/or measured and/or otherwise identified so that itspresence, absence, or a parameter or characteristic thereof can bemeasured and/or determined.

As used herein, the term “fluorescent label” refers to any molecule thatcan be detected via its inherent fluorescent properties, which includefluorescence detectable upon excitation. Suitable fluorescent labelsinclude, but are not limited to, fluorescein, rhodamine,tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins,pyrene, malachite green, stilbene derivatives, Lucifer yellow, CascadeBlue, Texas Red, IAEDANS, EDANS, boron-dipyrromethene (BODIPY), LC Red640, LC Red 705, cyanine dyes such as Cy3, Cy 5 and Cy 5.5, and Oregongreen, as well as to fluorescent derivatives thereof. Suitable opticaldyes are described in The Handbook: A Guide to Fluorescent Probes andLabeling Technologies. 2005, Haugland, R P. 10^(th) ed.Invitrogen/Molecular Probes; Carlsbad, Calif. Additional labels includebut are not limited to fluorescent proteins, such as green fluorescentprotein (GFP), yellow fluorescent protein (YFP), blue fluorescentprotein (BFP), cyan fluorescent protein (CFP) etc.

As used herein, a “protein tag” refers to a peptide sequence that isgenetically fused to a recombinant protein of interest. In general,protein tags are removable, for example by chemical agents or byenzymatic means. Protein tags may be used for facilitating purificationand/or proper folding of a recombinant protein of interest. Protein tagsare frequently referred to or classified according to their usage. Thusfor example, affinity tags are protein tags that enable purification ofthe recombinant protein to which they are fused using an affinitytechnique. Examples of affinity tags include, without limitation, chitinbinding protein (CBP), maltose binding protein (MBP), poly(His) andglutathione-S-transferase (GST). Solubilization tags assist in theproper folding in proteins and prevent their precipitation, such as forexample, thioredoxin (TRX) and poly(NANP). Chromatography tags alterchromatographic properties of the protein to afford different resolutionacross a particular separation technique, such as for example,polyanionic amino acids, such as FLAG-tag. Epitope tags are shortpeptide sequences which are capable of eliciting production ofhigh-affinity antibodies in many different species. Epitope tagsinclude, without limitation, V5-tag, c-myc-tag, and HA-tag, and can beused in antibody or antigen purification. Further, a protein tag may bea fluorescent protein, such as those described above.

The term “affinity tag” as used herein refers to any amino acid sequencefused to a protein of interest at either the amino terminal or carboxyterminal end of the protein. Typically, the affinity tag is used forisolation and or detection purposes. The “affinity tag” may optionallybe in the middle of the protein of interest such that when thecorresponding nucleic acid sequence is translated the affinity tag isfused in frame into the protein of interest. The amino acid residuesform a peptide that has affinity for a chemical moiety, a metal ion or aprotein. The affinity tag may have an overall positive, negative orneutral charge; typically the affinity tag has an overall positive ornegative charge.

The term “fusion protein” as used herein refers to a protein hybridcomprising amino acid sequence encoded by mRNA sequences fromheterologous proteins, such as for example a therapeutic protein and aprotein tag.

As used herein, the term “test compound” refers to a compound to betested by one or more screening assays of the invention as a putativeagent that modulates translation activity. The test compounds of theinvention encompass numerous classes of chemical molecules, thoughtypically they are organic molecules, and preferentially of lowmolecular weight. A drug candidate may be one type of test compound.

The term “modulator” as used herein is generic for an inhibitor oractivator of translation.

As used herein, the term “transcriptome” refers to the total set of allmessenger RNA (mRNA) molecules, or “transcripts”, produced in one or apopulation of cells, or within a particular subcellular compartment,tissue or organism. The transcriptome reflects the genes that are beingactively transcribed into mRNA at any given time, with the exception ofmRNA species that are degraded due to phenomena such as transcriptionalattenuation.

As used herein, the term “proteome” refers to the entire complement oftranslated proteins expressed by a cell, population of cells,subcellular compartment, tissue or organism. The proteome frequentlydoes not reflect the transcriptome of the corresponding cell,subcellular compartment, etc, since substantial regulation occurs duringtranslation, resulting in low correlation between a particular set ofmRNA transcripts existing at a particular time point and the set ofproteins that are actually translated from such available transcripts.The proteome is known to be dynamic and can change over time and/or inresponse to environmental conditions or exogenously added agents.

“Biotherapeutic agent,” as used herein, refers to a protein, enzyme,metabolite, nucleic acid, or microorganism that has therapeuticcharacteristics. Biotherapeutic agents originate from nature but can beengineered to produce optimal therapeutic value. The term includessynthetic mimics of naturally occurring proteins, enzymes, metabolites,nucleic acids, and microorganisms. Each possibility represents aseparate embodiment of the present invention.

The present invention provides systems for measuring and monitoringprotein translation of specified proteins in viable cells and inspecific subcellular compartments. The methods of the present inventionare capable of providing a signal indicating the rate of synthesis of aparticular protein of interest. The translation system of the presentinvention can be used to identify translation modulators inhigh-throughput screening (HTS).

The invention provides a system for measuring translation of a proteinof interest in a cell. The system comprises a cell, wherein the cellcomprises (i) a nucleic acid sequence encoding the protein of interest,and the nucleic acid is known to contain at least one predeterminedcodon pair or a codon pair of interest. The cell further comprises (ii)at least one tRNA pair labeled with a FRET pair. For any one tRNA pairin the system, each member of the tRNA pair is capable of reading onecodon of a predetermined codon pair in the nucleic acid. Further, tRNAspecies corresponding to those of the labeled tRNA pair occur inconsecutive order in the tRNA sequence of the protein of interest.

The codon pair may be selected on the basis of its encoding a dipeptideof interest. The predetermined codon pair may encode amino acids knownto occur in adjacent positions in the protein of interest. Referenceherein to amino acids occurring in adjacent positions means that theirconsecutive occurrence in the protein of interest may be in the orderfrom C-terminus to N-terminus, or in the order from N-terminus toC-terminus.

Opposite orientations of the dipeptide may occur in adjacent positionsin the amino acid sequence of the protein of interest. For example, thedipeptide arginine-phenylalanine (RF) may occur in opposite orientationsas RFFR. In a particular embodiment, at least one of the oppositeorientations of the dipeptide occurs in the amino acid sequence of theprotein of interest at a frequency higher than its frequency in theproteome of the cell or subcellular compartment under study. In aparticular embodiment, opposite orientations of the dipeptide occur inoverlapping positions in the amino acid sequence of the protein ofinterest. For example, the dipeptide arginine-phenylalanine (RF) mayoccur as RFR.

In a particular embodiment, opposite orientations of the dipeptide occurin overlapping positions in the tRNA sequence of the protein ofinterest. In a particular embodiment, the codons recognized by the tRNApair together correspond to an adjacent codon pair which occurs at aknown frequency within an mRNA transcript encoding the protein ofinterest. For example, the adjacent codon pair may be known to occurwithin the mRNA transcript at a frequency that is higher than thefrequency of the same codon pair in the transcriptome of the cell orsubcellular compartment.

The predetermined codon pair may be selected on the basis of itsfrequency i.e. relative occurrence, within the mRNA encoding theprotein. For example, in some preferred embodiments, the codon pairoccurs at a frequency within the mRNA that is relatively higher, ascompared to the frequency of the translation product of the same codonpair in the proteome of the cell, subcellular compartment, tissue ororganism under examination. Further, the frequency at which the codonpair occurs within the mRNA may be compared to the frequency of itstranslation product in the proteome of the same system, at a given timepoint.

In other particular embodiments, the frequency of the predeterminedcodon pair in the mRNA may be compared to the frequency of the samecodon pair in the transcriptome of the cell, subcellular compartmentthereof, tissue or organism under examination.

In some embodiments, it may be advantageous to conduct the comparison ofthe frequency of a codon pair relative to that of the proteome, ratherthan the transcriptome of the biological system under examination. It iswell documented that translational efficiency differs considerably forvarious eukaryotic mRNAs, thus leading to a significant statisticaldisparity between mRNA pools and the corresponding pool of proteinproducts. For example, structural features of mRNAs which have beendisclosed to influence their translational efficiency include the lengthof 5′ UTRs, and G+C content of 5′ UTRs (Kochetov et al., FEBS LEtt. 1998Dec. 4; 440(3):351-5). It has further been disclosed that upstream ORFsin mRNAs have a significant impact on translation of human mRNAs withmodest impact on mRNA levels (Calvo et al., Proc Natl Acad Sci U.S.A.2009 May 5; 106(18)7507-12).

In addition, the system of the invention may comprise a cell in which anucleic acid sequence of interest has been altered to increase thefrequency of a predetermined codon pair. In some cases, the nucleic acidsequence may be altered to decrease the frequency of a predeterminedcodon pair.

As used herein, the term “altered” refers to the process of changing anucleic acid sequence, for example, by site specific mutation so as toproduce a nucleic acid different from the wild type nucleic acid.Methods for gene alteration are well known in the art, as disclosed forexample in Sambrook et al (2000) “Molecular Cloning: A LaboratoryManual”, 3^(rd) ed. Cold Spring Harbor Laboratory Press.

In particular embodiments, opposite orientations of a predeterminedcodon pair occur in adjacent positions in the mRNA encoding the proteinof interest. For example, the codon pair CGT TTC, respectively encodingarginine-phenylalanine (RF), may appear in the mRNA sequence as CGT TTCTTC CGT. In a particular embodiment, at least one of the orientations ofthe predetermined codon pair occurs in the mRNA encoding the protein ofinterest at a frequency that is relatively higher, as compared to thefrequency of the same codon pair in the transcriptome of the cell.

The invention further involves use of at least one labeled tRNA pair.Each tRNA pair includes two tRNA members, of which each member maycorrespond to a single tRNA species. The relationship between the tRNApair and the predetermined codon pair is that the cognate amino acid ofeach tRNA member of the tRNA pair corresponds to one amino acid encodedby the predetermined codon pair.

Each member of the tRNA pair may be capable of reading the mostfrequently used codon on an mRNA molecule that encodes the cognate aminoacid of the tRNA pair member. In a particular embodiment, at least onemember of the tRNA pair is capable of reading the most frequently usedcodon on an mRNA molecule that encodes the cognate amino acid of thetRNA pair member.

The system may comprise a single tRNA pair, or a plurality of tRNApairs. In particular embodiments, the plurality of tRNA pairs comprisesthe complete set of isoacceptor tRNAs specific for one amino acidencoded by a predetermined codon pair. In other cases, the plurality oftRNA pairs comprises a subset of isoacceptor tRNAs specific for oneamino acid encoded by a predetermined codon pair.

The plurality of tRNA pairs may comprise tRNA species that have cognateamino acids that are different one from the other. However, a singletRNA pair or a plurality of tRNA pairs may be selected independently oftheir cognate amino acids.

In a particular embodiment, the plurality of tRNA pairs comprises afirst complete set of isoacceptor tRNAs specific for a first amino acidencoded by a predetermined codon pair, and further comprises a secondcomplete set of isoacceptor tRNAs specific for a second amino acidencoded by the same predetermined codon pair. In particular embodiments,each of the isoacceptor tRNAs of the first complete set may be labeledwith the same donor fluorophore, in which case each of the isoacceptortRNAs of the second complete set is labeled with the same acceptorfluorophore. Alternately, each of the isoacceptor tRNAs of the firstcomplete set may be labeled with the same acceptor fluorophore, in whichcase each of the isoacceptor tRNAs of the second complete set is labeledwith the same donor fluorophore.

However, in other embodiments, the first complete set of isoacceptorsmay be labeled with a plurality of different donor fluorophores, or witha plurality of different acceptor fluorophores, and similarly, in otherembodiments, the second complete set may be labeled with a plurality ofdifferent acceptor fluorophores, or with a plurality of different donorfluorophores. As is readily understood by one of skill in the art, thelabeling of the tRNAs is such that one set contains donor fluorophoresand the other set contains acceptor fluorophores, so that FRET signalsmay be produced when the appropriately labeled tRNA pairs are localizedin proximity within the same ribosomal complex.

In other embodiments, the plurality of tRNA pairs comprises a firstsubset of isoacceptor tRNAs specific for a first amino acid encoded bythe predetermined codon pair, and further comprises a second subset ofisoacceptor tRNAs specific for a second amino acid encoded by the samepredetermined codon pair. Accordingly, each of the isoacceptor tRNAs ofthe first subset may be labeled with the same donor fluorophore, inwhich case each of the isoacceptor tRNAs of the second subset is labeledwith the same acceptor fluorophore. Alternately, each of the isoacceptortRNAs of the first subset may labeled with the same acceptorfluorophore, in which case each of the isoacceptor tRNAs of the secondsubset are labeled with the same donor fluorophore.

However, in other embodiments, the first subset may be labeled with aplurality of different donor fluorophores, or with a plurality ofdifferent acceptor fluorophores. Similarly, the second subset may belabeled with a plurality of different donor fluorophores, or with aplurality of different acceptor fluorophores.

As is readily understood by one of skill in the art, the labeling of thetRNAs is such that one of the subsets contains donor fluorophores andthe other subset contains acceptor fluorophores, so that FRET signalsmay be produced when the appropriately labeled tRNA pairs are localizedin proximity within the same ribosomal complex.

In particular embodiments, the plurality of tRNA pairs comprises acomplete set of isoacceptor tRNAs specific for a first amino acidencoded by a predetermined codon pair, and further comprises a subset ofisoacceptor tRNAs specific for a second amino acid encoded by the samepredetermined codon pair. Each of the isoacceptor tRNAs of the completeset may be labeled with the same donor fluorophore, in which case eachof the isoacceptor tRNAs of the subset are labeled with the sameacceptor fluorophore. Alternately, each of the isoacceptor tRNAs of thecomplete set may be labeled with the same acceptor fluorophore, in whichcase each of the isoacceptor tRNAs of the subset are labeled with thesame donor fluorophore.

In a particular embodiment, the complete set is labeled with a pluralityof different donor fluorophores and the subset is labeled with aplurality of different acceptor fluorophores. In a particularembodiment, the complete set is labeled with a plurality of differentacceptor fluorophores and the subset is labeled with a plurality ofdifferent donor fluorophores.

The system may comprise a plurality of isoacceptor tRNAs specific forone amino acid encoded by a predetermined codon. In some cases, each ofthe isoacceptor tRNAs specific for one amino acid will be labeled withthe same donor fluorophore. In a particular embodiment, each of theisoacceptor tRNAs specific for one amino acid will be labeled with thesame acceptor fluorophore. In a particular embodiment, the plurality ofisoacceptor tRNAs specific for one amino acid will be labeled with aplurality of different acceptor fluorophores. In a particularembodiment, the plurality of isoacceptor tRNAs specific for one aminoacid will be labeled with a plurality of different donor fluorophores.

The system may comprise a first plurality of isoacceptor tRNAs specificfor a first amino acid that is encoded by one codon of a predeterminedcodon pair, and further comprise a second plurality of isoacceptor tRNAsspecific for a second amino acid that is encoded by the other codon ofthe same predetermined codon pair. The first plurality may be labeledwith the same donor fluorophore, in which case the second plurality islabeled with the same acceptor fluorophore. Alternately, the firstplurality may be labeled with the same acceptor fluorophore, in whichcase the second plurality is labeled with the same donor fluorophore.The first plurality of isoacceptor tRNAs may be labeled with a pluralityof different donor fluorophores, in which case the second plurality ofisoacceptor tRNAs is labeled with a plurality of different acceptorfluorophores. The first plurality of isoacceptor tRNAs may be labeledwith a plurality of different acceptor fluorophores, in which case thesecond plurality of isoacceptor tRNAs is labeled with a plurality ofdifferent donor fluorophores.

One member of any one tRNA pair can be capable of reading one codon ofeach of a plurality of predetermined codon pairs.

One member of any one tRNA pair can be capable of reading synonymouscodons encoding a particular amino acid.

The codons read by the tRNA pair together correspond to an adjacentcodon pair, and the occurrence of the adjacent codon pair within an mRNAtranscript encoding the protein of interest may be at a frequency thatis relatively higher, as compared to the frequency of the translationproduct of the same codon pair in the proteome of the cell or asubcellular compartment thereof. In a particular embodiment, thetranslation of the same codon pair in the proteome is at a given timepoint.

In other particular embodiments, the adjacent codon pair occurs withinan mRNA transcript encoding the protein of interest at a frequency thatis relatively higher, as compared to the frequency of the same adjacentcodon pair in the transcriptome of the cell or a subcellular compartmentthereof.

The system may comprise two or more tRNA pairs labeled with two or moreFRET pairs. In a particular embodiment, the system comprises two tRNApairs, and each of the codon pairs translated by the respective tRNApairs occurs at a frequency in the mRNA encoding the protein that isrelatively higher, as compared to the frequency of the translationproduct of the same codon pairs in the proteome of the cell. In aparticular embodiment, the frequency comparison relative to the proteomeof the cell is that determined at a given time point.

In a particular embodiment, the system comprises two tRNA pairs, andeach of the codon pairs translated by the respective tRNA pairs occursat a frequency in the mRNA encoding the protein that is relativelyhigher, as compared to the frequency of the same codon pairs in thetranscriptome of the cell.

The system may comprise two different tRNA pairs, wherein each tRNA paircorresponds to a distinct codon pair, and each distinct codon pairoccurs in one of two different mRNAs which respectively encode twodifferent proteins of interest. The system may comprise two differenttRNA pairs and two different mRNAs, the latter of which respectivelyencode two different proteins of interest. In this embodiment, each tRNApair corresponds to a distinct codon pair within one of the twodifferent mRNAs.

In the system comprising two different tRNA pairs and two differentmRNAs, each codon pair may occur in its respective mRNA at a frequencythat is relatively higher, as compared to the occurrence of thetranslation product of the same codon pair in the proteome of the cell.The frequency comparison relative to the proteome of the cell may bethat determined at a given time point.

In a particular embodiment, one tRNA pair corresponds to a codon pairwhich occurs in its respective mRNA at a frequency that is relativelyhigher, as compared to the frequency of the translation product of thesame codon pair in the proteome of the cell. In a particular embodiment,the frequency of the translation product of the same codon pair in theproteome is that determined at a given time point.

According to a particular embodiment, the present invention provides asystem for measuring translation of a predetermined tRNA pair sequencewithin a protein of interest, the system comprising a cell orsubcellular compartment, wherein the cell or subcellular compartmentcomprises a nucleic acid sequence encoding the protein of interest, andwherein the cell further comprises a first tRNA labeled with a donorfluorophore and a second tRNA labeled with an acceptor fluorophore,wherein donor and acceptor fluorophores from a FRET pair In oneembodiment, the cell being assayed is an intact cell.

In particular embodiments, the system is utilized for measuring thelevel of translation of the tRNA pair of interest; subsequently,proteins of interest whose tRNA sequence contains the tRNA pair may beidentified. In another embodiment, translation of one or more particularproteins of interest whose tRNA sequence contains the tRNA pair isassessed.

In another embodiment, the present invention provides a system formeasuring translation of a protein of interest, the system comprising asubcellular compartment, wherein the subcellular compartment comprises(i) an mRNA transcript encoding the protein of interest, and (ii) afirst tRNA labeled with a donor fluorophore and a second tRNA labeledwith an acceptor fluorophore, and wherein the codon pair recognized bythe first and second tRNA occur adjacently in the codon sequence of themRNA transcript at a frequency higher than the frequency of the samecodon pair in the transcriptome of the subcellular compartment.

In a particular embodiment, the tRNA pair defined by the first andsecond tRNA occur in the tRNA sequence of the protein of interest at afrequency higher than the frequency of the translation product of thesame tRNA pair in the proteome of the cell. Thus, the system is capableof producing a FRET signal that is enriched during translation of thetranscript of interest, relative to general translation in thesubcellular compartment being assayed.

In another embodiment, the adjacent occurrence of the codons read by thefirst and second tRNA is enriched in the transcript of interest,relative to the transcriptome of the subcellular compartment.

In another embodiment, the invention provides a system for measuringtranslation of a protein of interest, the system comprising asubcellular compartment, wherein the subcellular compartment comprises(i) a nucleic acid sequence encoding the protein of interest; and (ii) afirst tRNA labeled with a donor fluorophore and a second tRNA labeledwith an acceptor fluorophore, and wherein the tRNA pair defined by theconsecutive order of the first tRNA and the second tRNA occurs in thetRNA sequence of the protein of interest at a frequency higher than thefrequency of the translation product of the same tRNA pair in theproteome of that sub cellular compartment

Reference herein to a “tRNA labeled with a donor fluorophore” or to a“tRNA labeled with a acceptor fluorophore” refers to tRNA moleculesdirectly labeled with the specified fluorophore, and is distinct fromtRNA molecules acylated with an amino acid analogue containing afluorophore.

In particular embodiments of methods and systems of the presentinvention, a nucleic acid sequence of the gene encoding the protein inthe cell or subcellular compartment has been altered so as to increasethe frequency of the tRNA pair in the corresponding tRNA sequence of theprotein. In another embodiment, a method of the present inventionfurther comprises the step of engineering a gene encoding a protein ofinterest to increase the frequency of adjacent codons that are read bythe tRNA pair of interest.

In another embodiment, the present invention provides a system formeasuring utilization of a tRNA pair of interest in protein synthesis,the system comprising a cell or subcellular compartment, wherein thecell or subcellular compartment comprises (i) an mRNA encoding a proteinof interest, and (ii) a first tRNA labeled with a donor fluorophore anda second tRNA labeled with an acceptor fluorophore, and wherein thefirst and second tRNA together form the tRNA pair of interest. In oneembodiment, the cell being assayed is an intact cell.

In another embodiment of methods and systems of the present invention,the tRNA pair whose utilization is measured recognize codons which occuradjacently in an mRNA encoding a protein of interest, and this adjacentcodon pair occurs in the mRNA encoding the protein of interest at afrequency that is relatively higher, as compared to the frequency of thesame codon pair in the transcriptome of the cell or subcellularcompartment being assayed. Thus, the FRET signal is enriched duringtranslation of the protein of interest, relative to the signal producedby general translation in the cell being assayed.

A frequency of a codon pair or a translation product thereof, or of atRNA pair, that is denoted as “significantly higher” or “relativelyhigher” in comparison to a reference frequency of the same pair in theproteome or transcriptome of the cell or subcellular compartment refers,in particular embodiments, to a frequency that is at least two-foldhigher than the frequency in the reference proteome or transcriptome; orat least three-fold higher; or least four-fold higher; or at leastfive-fold higher; or least six-fold higher; or at least eight-foldhigher; or at least 10-fold higher; or at least 15-fold higher; or atleast 20-fold higher; or at least 30-fold higher; or at least 50-foldhigher; or at least 100-fold higher.

A non-limiting example of a tRNA pair enriched in a protein of interestis the tRNA pair where the first tRNA is capable of being charged withthe amino acid Methionine (M) and the second with the amino acidTryptophane (W). There is a single tRNA species for M and a single tRNAfor W. This dipeptide is about 16-fold more abundant in the tRNAsequence of proinsulin than in the human proteome, and the correspondingcodon pair is similarly more abundant in the transcript encodingproinsulin than in the human transcriptome. Each possibility representsa separate embodiment of the present invention.

By way of example, once a cell has been labeled according to theprinciples of the invention to monitor production of a protein ofinterest, it is possible to compute the confidence of the resultingassay. That is, upon detection and measurement of a FRET signal, it ispossible to determine the confidence that the protein of interest isindeed being translated. This requires (a) an estimate of the enrichmentE of the selected tRNA pair compared to the average, or backgroundproteins; and (b) an estimate of the fraction f of the rate ofproduction of the protein of interest out of the entire proteinproduction in that cell or tissue. For example, in the case of insulin,we have E=16 and we estimate f=0.25.

To compute the confidence, the tRNA pair of choice is assumed to appearat a frequency f_(d) in the background proteins; therefore the rate ofFRET signal generation by background protein production is proportionalto f_(d)*(1−f), and the rate of FRET signal generation by production ofthe protein of choice is proportional to E*f_(d)*f. Therefore theprobability that an observed signal is due to production of the proteinof choice is E*f_(d)*f/(E*f_(d)*f+f_(d)*(1−f))=Ef/(Ef+1−f). In the caseof insulin, this becomes 16*0.25/(16*0.25+0.1)=4/4.1=97.5% confidencethat insulin is being produced.

Estimation of the enrichment factor E can be performed in several ways.E is computed by comparing the rate of appearance of a specific tRNApair in a protein of choice with its appearance in all other proteinsproduced by a cell or tissue. The rate of appearance of a specific tRNApair in a protein of interest can be computed from the protein sequence.To analyze this rate in the proteome of a cell, one of severalstrategies can be employed. First, given the genome of that organism,the entire genome can be used as an estimation of the proteins producedby the cell. If additional information is available, for example ifproteins specifically produced by that cell type or tissue are known,than this information can be used in addition or alternately. Finally,the average rate of FRET production by that tRNA pair can be measureddirectly, with and without the protein of interest, such as in caseswhere the protein of interest is being transfected into cells.

These analyses can be performed either by analyzing amino acid sequencesor tRNA sequences, to obtain an optimal labeling strategy either withall isoaccepting tRNAs of a given amino acid or by single moiety tRNAs.

In addition, the sequence of proteins to be produced can sometimes beedited or altered to achieve various benefits. If such edits arepermissible, a specific motif can be inserted into the sequence toensure high confidence identification of the production of the proteinof interest. For example, the amino acid pairs WW and CW occur veryrarely in the mouse proteome. A poly-W or poly-CW tail or tag engineeredto be expressed within or at a terminus of a protein of interest willensure confident identification of production of this protein in WWlabeled murine cells and tissues. For purposes of illustration,calculations are provided herein for several proteins of interest. Inone example, filgrastim is a recombinant human G-CSF (Granulocyte colonystimulating factor), marketed by Amgen under the brand name Neupogen™.This is a 175 residue recombinant protein produced in E. coli.

Analysis of the amino acid sequence of filgrastim and the sequences ofall E. coli protein sequences shows that the amino acid pairs CL and LC(Cysteine and Leucine) appear a total of 4 times in the sequence of thetarget protein (filgrastim) and 3216 times in the background (entireamino acid sequence of E. coli proteins, which includes a total of1,328,825 dipeptides). Computation of the enrichment factor E yields

E(C,L)=(4/174)/(3216/1,328,825)=9.50.

In another example, somatotropin is a recombinant human growth hormonewith 190-191 residues, marketed by various vendors under several brandnames. It is synthesized in E. coli. Analysis of the amino acid sequenceof somatotropin and the sequences of all E. coli protein sequences showsthat the amino acid pairs CF and FC (Cysteine and Phenylalanine) appeara total of 2 times in the sequence of the target protein (somatotropin)and 1388 times in the background. Computation of the enrichment factoryields

E(C,F)=(2/190)/(1388/1,328,825)=10.08.

In another example, human serum alanine aminotransferase (ALT) isregarded as an indicator of liver damage based on the presumption thatALT protein is specifically and abundantly expressed in the liver. ALT 1and ALT 2 are two homologous isoforms with 70% identity and 85%similarity. Analysis of the tRNA sequences of ALT 1, ALT 2 and the humangenome considered as a background or average protein identifies severaltRNAs pairs that are specific to ALT 1 but not ALT 2 or background, andseveral tRNAs pairs that are specific to ALT 2 but not Alt1 orbackground, as shown in Table 2. In addition, numerous tRNA pairs out ofthe total of 1225 possible tRNA pairs can be found which are representedin the background but not in either ALT 1 or ALT 2. This exampleillustrates the ability to use tRNA pairs for simultaneously monitoringmore than one protein and distinguishing these proteins from each otherand from the background, even in case of significant homology.

TABLE 2 Frequency in Frequency Frequency Enrichment tRNA pair backgroundin Alt1 in Alt2 factor Arg4Ala2 0.000131 0.004032 0 30.81001 Pro2Arg10.000271 0.004032 0 14.90603 Tyr2Ser2 0.000147 0.002016 0 13.72198Arg2Arg2 0.000308 0 0.005736 18.60601 Ala2Tyr2 0.000133 0 0.00191214.37869 Arg2Ser3 0.000138 0 0.001912 13.83889

In another example, etanercept is a dimeric fusion protein consisting ofthe extracellular ligand-binding portion of the human 75 kiloDalton(p75) tumor necrosis factor receptor (TNFR) linked to the Fc portion ofhuman IgG1. Etanercept is produced by recombinant DNA technology in aChinese hamster ovary (CHO) mammalian cell expression system. Itconsists of 467 amino acids. It is marketed by Immunex under the brandname of Enbrel™. To determine the best labeling strategy, the mousegenome was used as an approximation to the unavailable proteome of CHOcells, since the Chinese Hamster and mouse have a very similar genomicstructure. Analysis of the amino acid sequence of etanercept and thesequences of all mouse protein sequences shows that the amino acid pairsWN and NW (Asparagine and Tryptophan) appear a total of 4 times in thesequence of the target protein and 8313 times in the background.

Computation of the enrichment factor E yields:

E(W,N)=(4/466)/(11644/11,638,331)=8.58.

In another example, cetuximab is an epidermal growth factor receptorbinding FAB. Cetuximab is composed of the Fv (variable; antigen-binding)regions of the 225 murine EGFr monoclonal antibody specific for theN-terminal portion of human EGFr with human IgG1 heavy and kappa lightchain constant (framework) regions. It is marketed by ImClone™ systemsunder the brand name of Erbitux™. To determine the best labelingstrategy, the mouse genome was used as an approximation to theunavailable proteome of CHO cells. Analysis of the 452-long amino acidsequence of heavy-chain I of cetuximab and the sequences of all mouseprotein sequences shows that the amino acid pairs WY and YW (Tyrosineand Tryptophan) appear a total of 2 times in the sequence of the targetprotein and 17296 times in the background. Computation of the enrichmentfactor E yields

E(Y,W)=(2/451)/(8313/11,638,331)=6.21.

In another embodiment, comparison of the sequence of the specificprotein to the sequences of background proteins is performed on thebasis of adjacent tRNA pairs. In this analysis, two histograms areprepared of all consecutive pairs of tRNAs in the protein of interestand in all background proteins. In this histogram, directionality isdisregarded; that is, R₅L₃ and L₃R₅ are considered identical. In Homosapiens, there are 49 distinct tRNAs (including selenocysteine), so thatthis histogram contains (49*49+49)/2=1225 entries. This is reached asfollows: (49*49-49)/2 is the number of hetero-tRNA-pairs, and 49 is thenumber of homo-tRNA-pairs (both member have the same species, where onemember is labeled with the donor and the other with the acceptorfluorophore of the FRET pair). The number of possible pairs is thus

(49*49−49)/2+49=(49*49+49)/2=49*50/2=1225.

In another embodiment, comparison of the sequence of the specificprotein to the sequences of background proteins is performed on thebasis of adjacent amino acid pairs. In this analysis, two histograms areprepared of all consecutive pairs of amino acids in the protein ofinterest and in all background proteins. In this histogram,directionality is disregarded; that is, FY and YF are consideredidentical. This histogram contains (20*20+20)/2=210 entries. This isreached as follows: (20*20-20)/2 is the number of hetero-dipeptides, and20 is the number of homo-dipeptides (peptides can be labeled both asdonor and acceptor). The number of possible pairs is thus(20*20-20)/2+20=(20*20+20)/2.

After this histogram is determined, it is normalized so that the sum ofall its entries becomes equal to unity. Then, the two histograms arecompared by dividing the value of the entry in the histogram for theprotein of interest by the corresponding entry for the backgroundproteins. The entry provides the significance value for that amino acidpair. The entry with the highest factor is a leading candidate for usein a method of the present invention. The assay herein disclosed can befurther enhanced by using more than one tRNA pair, either simultaneouslyin the same cell or cells (by using distinct spectral properties of thevarious pairs) and/or separately, for example in different wells of amulti-well assay plate. In this way, if a single tRNA pair provides anassay for production of a protein of interest with confidence of C1, andif another assay with a distinct pair provides a confidence of C2, thanthe combination of these two assay provides confidence of1−(1−C1)*(1−C2). Clearly, this concept can be generalized up to themaximal number of bi-markers, 210 pairs for the case of amino-acidisoaccepting tRNA sets, or N*(N+1)/2 for N tRNA moieties, where N=49 forHomo sapiens cells, N=20 for Homo sapiens mitochondria and so on foreach case.

In various embodiments, a cell or subcellular compartment of a system ofthe present invention further comprises a second tRNA pair labeled witha second FRET pair i.e. a third tRNA labeled with a second donorfluorophore and a fourth tRNA labeled with a second acceptor fluorophore(collectively termed a “second tRNA pair labeled with a second FRETpair”). Either or both of the first and second tRNA pairs may correspondto pairs of adjacent tRNAs which occur in the tRNA sequence of theprotein of interest at a frequency significantly higher than thefrequency of the same pair in the proteome of the cell or subcellularcompartment. The third and fourth tRNA thus produce a second FRET signaldistinct from the FRET signal produced by the first and second tRNA ofthe first tRNA pair. In this embodiment, the emission spectrum of thesecond donor fluorophore overlaps with the excitation spectrum of thesecond acceptor fluorophore. In this embodiment, the second FRET signalreflects general translation in the cell or subcellular compartmentbeing assayed, not including translation of the transcript of interest.In another embodiment, translation of the transcript of interest makes arelatively small contribution to the second FRET signal which can becomputed and factored out. In either case, analysis of the first andsecond FRET signals enables accurate determination of the level oftranslation of the protein of interest.

In another embodiment, the frequency of the adjacent codonscorresponding to the third and fourth tRNA is significantly lower in thetranscript of interest, relative to the transcriptome of the cell orsubcellular compartment. Each possibility represents a separateembodiment of the present invention.

In another embodiment, a system of the present invention furthercomprises an additional cell, the additional cell comprising a secondtRNA pair labeled with a second FRET pair, wherein the second tRNA paircorresponds to a pair of adjacent tRNAs which occurs in the tRNAsequence of the protein of interest at a frequency significantly higherthan the frequency of the translation product of the same pair in theproteome of the cell.

In another embodiment, the additional cell is substantially identical tothe cell containing the first tRNA pair. The second FRET pair comprisesa second donor fluorophore and a second acceptor fluorophore, whereinthe emission spectrum of the second donor fluorophore overlaps with theexcitation spectrum of the second acceptor fluorophore. In thisembodiment, the FRET signal from the second tRNA pair reflects generaltranslation in the cell being assayed, not including translation of thetranscript of interest. In another embodiment, the adjacent occurrenceof codons corresponding to the third and fourth tRNA is significantlylower in the transcript of interest, relative to the transcriptome ofthe cell. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, a system of the present invention furthercomprises an additional subcellular compartment, the additionalsubcellular compartment comprising a second tRNA pair labeled with asecond FRET pair, wherein the second tRNA pair corresponds to a pair ofadjacent tRNAs which occurs in the tRNA sequence of the protein ofinterest at a frequency significantly higher than the frequency of thetranslation product of the same pair in the proteome of the subcellularcompartment. In another embodiment, the additional subcellularcompartment is substantially identical to the subcellular compartmentcontaining the first tRNA pair. The second FRET pair comprises a seconddonor fluorophore and a second acceptor fluorophore, wherein theemission spectrum of the second donor fluorophore overlaps with theexcitation spectrum of the second acceptor fluorophore. In thisembodiment, the FRET signal from the second tRNA pair reflects generaltranslation in the subcellular compartment being assayed, not includingtranslation of the transcript of interest.

In another embodiment, the frequency in the transcript of interest of acodon pair corresponding to the first tRNA pair is significantly lowerin said transcript, relative to the frequency of the same codon pair inthe transcriptome of the subcellular compartment.

In another embodiment, FRET signals emitted from the first and secondlabeled tRNA pairs (also referred to respectively as the “first” and“second FRET signals”) of the present invention are capable of beinganalyzed together in order to increase the accuracy of measurement oftranslation of a transcript of interest. In another embodiment, analysisof the first and second FRET signals together enables more accuratedetermination of the contribution of background translation (translationof transcripts other than the transcript of interest), therebyincreasing the signal-to-noise ratio of measurement of the transcript ofinterest. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment of methods and systems of the present invention,the sequence of the gene encoding the protein in the cell or subcellularcompartment has been altered to increase the frequency of the codon paircorresponding to the third and fourth tRNA pair.

It will be understood by those of skill in the art that, in an instancewherein the third and fourth tRNAs introduced to a sample that isdifferent from the sample containing the first and second tRNAs, thesecond donor fluorophore may be the same as or different from the firstdonor fluorophore of the present invention. In an instance wherein thefirst, second, third, and fourth tRNAs are all present in the samebiological sample, the second donor fluorophore must have an excitationor emission spectrum distinct from that of the first donor fluorophore.In another embodiment, the emission or excitation spectra of the firstand second donor fluorophores can overlap, but must not be identical,and it should preferably be possible to separate their contributions toFRET signals generated by the biological sample. Each possibilityrepresents a separate embodiment of the present invention.

Similarly, it will be understood that, in an instance wherein the thirdand fourth tRNAs (i.e. the second tRNA pair) are present in a sampledifferent from the sample containing the first and second tRNAs (i.e.the first tRNA pair), the second acceptor fluorophore may be the same asor different from the first acceptor fluorophore of the presentinvention. In an instance wherein the first, second, third, and fourthtRNAs are all present in the same biological sample, the second acceptorfluorophore must have an excitation or absorption spectrum distinct fromthe first acceptor fluorophore. In another embodiment, the excitationand absorption spectra of the first and second acceptor fluorophores canoverlap, but must not be identical, and in preferable embodiments, theircontributions to FRET signals generated by the biological sample aredistinguishable and separated.

In another embodiment, a plurality of cells or subcellular compartmentscomprising the labeled element is utilized in a system of the presentinvention.

In another embodiment, the present invention provides a method forsorting a cell population based on its expression level of a protein,the method comprising the steps of performing a method of the presentinvention, measuring the signals produced thereby, and sorting thecells.

In another embodiment, the present invention provides a method forenriching a cell population based on its expression level of a protein,the method comprising the steps of performing a method of the presentinvention, measuring the signals produced thereby, and enriching forthose cells with increased expression of the protein of interest.

In another embodiment, the present invention provides a method forscreening cell populations based on their expression levels of aprotein, the method comprising the steps of performing a method of thepresent invention, measuring the signals produced thereby, andidentifying cell populations with increased expression of the protein ofinterest. In another embodiment, the cell populations are clonalpopulations. In another embodiment, the method is a high-throughputscreening method. Each possibility represents a separate embodiment ofthe present invention.

In another embodiment, the system of the present invention furthercomprises instructions for use thereof in measuring or monitoringtranslation of a protein of interest in an intact cell or a cellularorganelle.

In another embodiment, the invention provides a method for measuringtranslation of a protein of interest in a biological sample comprisingat least one cell, the method comprising the steps of:

-   -   (vi) selecting a specific tRNA pair which occurs in the tRNA        sequence of a protein of interest at a known frequency;    -   (vii) introducing into the biological sample a tRNA pair labeled        as a FRET pair, wherein an acceptor fluorophore and a donor        fluorophore together form the FRET pair and wherein each member        of the tRNA pair is labeled with one of the acceptor fluorophore        or the donor fluorophore, and wherein the tRNA pair corresponds        to a consecutive pair of codons in the mRNA of the protein of        interest; and    -   (viii) detecting FRET signals emitted from the biological sample        during protein translation, thereby measuring translation of the        protein of interest.

The frequency of the tRNA pair in the tRNA sequence of a protein may beknown to be higher or lower than the frequency of its correspondingtranslation product in the proteome of the cells of the biologicalsample. In a particular embodiment, a second tRNA pair is introduced tothe sample. The second tRNA pair may be introduced into a cell orsubcellular compartment that is other than that into which the firsttRNA pair is introduced. In the case of either or both of the first tRNApair and second tRNA pair, opposite orientations of the particular pairmay occur in adjacent positions in the tRNA sequence of the protein ofinterest. In the case of either or both of the first tRNA pair andsecond tRNA pair, opposite orientations of the particular pair may occurin overlapping positions in the tRNA sequence of the protein ofinterest.

There is further provided a method for measuring translation of aprotein of interest in a biological sample comprising at least one cell,the method comprising the steps of:

-   -   (i) selecting a first codon pair which occurs in the mRNA        sequence encoding a protein of interest at a known frequency;    -   (ii) introducing into the biological sample comprising cells at        least one first tRNA pair, wherein for each first tRNA pair,        each tRNA member of the tRNA pair is capable of reading one        codon of the first codon pair selected in (i); and wherein one        tRNA member of the tRNA pair is labeled with a donor fluorophore        and the other member of the tRNA pair is labeled with an        acceptor fluorophore, and wherein the donor fluorophore and the        acceptor fluorophore together form a FRET pair; and    -   (iii) detecting FRET signals emitted from the tRNA pair during        protein translation, thereby measuring translation of the        protein of interest.

The codon pair selected in (i) may occur in the mRNA sequence at afrequency that is relatively higher, as compared to the frequency of thetranslation product of the same codon pair in the proteome of the cellor subcellular compartment thereof comprising the biological sample. Inparticular embodiments, the frequency of said translation product in theproteome is that determined at a given time point.

In a particular embodiment, the method further comprises a step ofaltering the nucleic acid sequence of the gene encoding the protein ofinterest so as to increase the frequency of the codon pair in the mRNAsequence encoding the protein.

In a particular embodiment, the method further comprises:

-   -   (ix) selecting a second codon pair, wherein the second codon        pair occurs in the mRNA sequence encoding the protein of        interest at a relatively higher frequency as compared to the        frequency of the translation product of the second codon pair in        the proteome of the biological sample; and    -   (x) introducing into the biological sample at least one second        tRNA pair, wherein each tRNA member of the second tRNA pair is        capable of reading one codon of the second codon pair selected        in (iv), and wherein each tRNA member of the second tRNA pair is        labeled with either a second donor fluorophore or a second        acceptor fluorophore, and wherein the second donor fluorophore        and the second acceptor fluorophore together form a second FRET        pair.

In a particular embodiment, at least one of the acceptor fluorophore andthe donor fluorophore of the second FRET pair is other than that of thefirst FRET pair. In a particular embodiment, the acceptor fluorophoreand the donor fluorophore of the second FRET pair are both other thanthat of the first FRET pair.

In a particular embodiment, the method further comprises altering anucleic acid sequence of the gene encoding the protein so as to increasethe frequency of the second codon pair in the mRNA sequence encoding theprotein of interest.

In a particular embodiment, opposite orientations of the second codonpair occur in adjacent positions in the mRNA sequence encoding theprotein of interest.

The introducing of the second tRNA pair in (v) may be into a cell orsubcellular compartment that is other than that of the introducing ofthe first tRNA pair in (ii).

In one embodiment, the codon pair selected in (i) is one which occurs inthe mRNA sequence of the protein of interest at a frequency that isrelatively higher, as compared to the frequency of the translationproduct of the same codon pair in the proteome of a subcellularcompartment of the biological sample, and the introducing of the tRNApair in (ii) is into the subcellular compartment.

The biological sample may be selected from the group consisting of acell line, a primary cell culture, a whole organism or a subcellularcompartment thereof. The step of introducing can be performed in cells,such as mammalian cells, vertebrate cells, avian cells, insect cells,yeast cells and plant cells. The mammalian cells may be human cells ornon-human mammalian cells. In a particular embodiment, the biologicalsample comprises diseased cells. In a particular embodiment, the step ofintroducing is into a subcellular compartment of living cells. Thesubcellular compartment may be selected from dendritic spines,mitochondria, endoplasmic reticulum (ER) and chloroplasts.

The whole organism may be a model organism used for genetic research,such as Escherichia. coli, Caenorhabditis elegans, Drosophilamelanogaster, Arabidopsis thaliana, and an inbred strain of Musmusculus. The model organism may be any other model organism used forgenetic research.

The cells used in the system and methods of the invention may be live orfixated cells. Fixated cells may additionally be permeabilized. Methodsfor chemically fixating cells are well known in the art and include ingeneral, use of organic solvents and/or cross-linking reagents. Organicsolvents used for fixation include various alcohols, acetone and aceticacid. Cross-linking reagents used for fixation include variousaldehydes, such as formalin and paraformaldehyde. Cross-linking reagentsform intermolecular bridges and thus may be preferable over certainorganic solvents for preserving cell structure. Other agents used forchemical fixation include for example, oxidizing agents, mercurialcompounds, picrates, HOPE fixative (Hepes-glutamic acid buffer-mediatedorganic solvent protection effect)

According to other embodiments, the biological sample comprisesmitochondria and at least one of said first tRNA and said second tRNA isa mitochondria-specific tRNA. In other embodiments, both said first tRNAand said second tRNA are a mitochondria-specific tRNA. Each possibilityrepresents a separate embodiment of the present invention.

According to other embodiments, the step of introducing is intomitochondria and the tRNA pair comprises at least onemitochondria-specific tRNA. In other embodiments, both members of thetRNA pair are mitochondria-specific tRNA.

In a particular embodiment, the method further comprises a step ofirradiating the biological sample with electromagnetic radiation priorto the step of measuring the emitted electromagnetic radiation, whereinthe electromagnetic radiation used for irradiating is of a wavelengthdifferent from the detected FRET signals.

In a particular embodiment, the method further comprises a step ofaltering the nucleic acid sequence of the gene encoding the protein ofinterest so as to increase the frequency of the tRNA pair in the tRNAsequence of the protein.

In another embodiment, the present invention provides a method formeasuring translation of a transcript of interest in a cell, the methodcomprising the steps of:

-   -   (iii) introducing into a cell a first tRNA and a second tRNA,        wherein the first tRNA is labeled with a donor fluorophore, and        the second tRNA is labeled with an acceptor fluorophore, wherein        the cell comprises the transcript of interest, and wherein the        adjacent codon pair recognized by the first tRNA and the second        tRNA occurs in the codon sequence of the transcript of interest        at a frequency significantly higher than the frequency of the        same codon pair in the transcriptome of the cell; and    -   (iv) detecting FRET signals emitted from the cell.

In another embodiment, the present invention provides a method formeasuring translation of a transcript of interest in a subcellularcompartment, the method comprising the steps of:

-   -   (iii) introducing into a subcellular compartment a first tRNA        and a second tRNA, wherein the first tRNA is labeled with a        donor fluorophore, and the second tRNA is labeled with an        acceptor fluorophore, wherein the subcellular compartment        comprises the transcript of interest, and wherein the adjacent        codon pair recognized by the first tRNA and the second tRNA        occurs in the codon sequence of the transcript of interest at a        frequency significantly higher than the frequency of the same        codon pair in the transcriptome of the subcellular compartment;        and    -   (iv) detecting FRET signals emitted from the subcellular        compartment.

In another embodiment, the adjacent occurrence of the first and secondtRNA is enriched in the transcript of interest, relative to thetranscriptome of the subcellular compartment. In another embodiment, thesubcellular compartment is an organelle.

In other embodiments, a method of the present invention is used tomeasure translation of a protein of interest in a cell or subcellularcompartment. In this embodiment, the first tRNA and second tRNA of thepresent invention are present in the tRNA sequence of the protein ofinterest at a frequency significantly higher than the frequency of thecorresponding codon in the transcriptome of the cell or subcellularcompartment; or at a frequency significantly higher than the frequencyof the corresponding translation product in the proteome. Eachpossibility represents a separate embodiment of the present invention.

In one embodiment, the method for measuring translation of a protein ofinterest in a biological sample comprises the steps of:

-   -   (iii) selecting one or more tRNA pairs which occurs in the tRNA        sequence of the protein of interest at a known frequency,        wherein each tRNA pair is defined by a first set of tRNA and a        second set of tRNA;    -   (iv) introducing into a biological sample the first set of tRNA        and the second set of tRNA, wherein the tRNA in the first set        are labeled with a donor fluorophore, and the tRNA in the second        set are labeled with an acceptor fluorophore, and wherein the        biological sample further comprises a nucleic acid sequence        encoding the protein of interest, and wherein the frequency of        the occurrence of the tRNA pair in the tRNA sequence of the        protein of interest is higher than the frequency of the        translation product of the tRNA pair in the proteome of the        cell; and    -   (iii) detecting FRET signals radiation emitted from the        biological sample, thereby measuring translation of the protein        of interest.

In another embodiment, the present invention provides a method formeasuring the effect of a drug candidate on translation of a transcriptof interest, the method comprising the steps of:

-   -   (vi) introducing into a intact cell a first tRNA and a second        tRNA, wherein the first tRNA and second tRNA are labeled with a        donor fluorophore, and an acceptor fluorophore, respectively,        wherein the cell comprises the transcript of interest, and        wherein the adjacent codon pair recognized by the first and        second tRNA occurs gain the codon sequence of the transcript of        interest at a frequency significantly higher than the frequency        of the translation product of the same codon pair in the        proteome of the cell;    -   (vii) measuring electromagnetic radiation emitted from the        acceptor fluorophore;    -   (viii) contacting the cell with the drug candidate; and    -   (ix) measuring electromagnetic radiation emitted from the cell,        under the conditions of (iii), whereby a significant difference        between the electromagnetic radiation measured in step (ii) and        the electromagnetic radiation measured in step (iv) indicates        that the drug candidate affects the translation of a transcript        of interest.

Step (ii) is performed under conditions wherein the cell has not beencontacted with the drug candidate. It will be understood to those ofskill in the art that the cell can comprise the transcript of interestby virtue either of the presence therein of DNA that encodes thetranscript of interest or by transfection of the transcript of interestinto the cell. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the present invention provides a method formeasuring the effect of a drug candidate on translation of a transcriptof interest, the method comprising the steps of:

-   -   (iv) introducing into a subcellular compartment a first tRNA and        a second tRNA, wherein the first tRNA and second tRNA are        labeled with a donor fluorophore, and an acceptor fluorophore,        respectively, wherein the subcellular compartment comprises the        transcript of interest, and the adjacent codon pair recognized        by the first and second tRNA occurs in the codon sequence of the        transcript of interest at a frequency significantly higher than        the frequency of the translation product of the same codon pair        in the proteome of the subcellular compartment;    -   (v) measuring electromagnetic radiation emitted from the        subcellular compartment;    -   (vi) contacting the subcellular compartment with the drug        candidate; and    -   (x) measuring electromagnetic radiation emitted from the        subcellular compartment, under the conditions of (iii), whereby        a significant difference between the electromagnetic radiation        measured in step (ii) and the electromagnetic radiation measured        in step (iv) indicates that the drug candidate affects the        translation of a transcript of interest.

Step (ii) is performed under conditions wherein the subcellularcompartment has not been contacted with the drug candidate. It will beunderstood to those of skill in the art that the subcellular compartmentcan comprise the transcript of interest by virtue either of the presencetherein of DNA that encodes the transcript of interest or bytransfection of the transcript of interest into the subcellularcompartment. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, step (iv) of one of the above methods isperformed immediately after administration of the drug candidate. Inanother embodiment, an equilibration period of 5-120 minutes is allowedprior to the translation assay. In another embodiment, step (iv) isperformed at a time wherein the drug candidate is still present ineffective quantities. In another embodiment, step (iv) is performed at atime wherein the first and second labeled tRNA are still present inquantities substantially equivalent to those in step (ii). In anotherembodiment, the first and second labeled tRNA are re-administered to thebiological sample in order to be present in quantities substantiallyequivalent to those in step (ii). Each possibility represents a separateembodiment of the present invention.

In another embodiment, the present invention provides a method formeasuring the effect of a drug candidate on translation of a transcriptof interest, the method comprising the steps of:

-   -   (v) introducing into each of a first cell preparation and a        second cell preparation a first tRNA and a second tRNA, wherein        the first and second tRNA are labeled with a donor fluorophore        and an acceptor fluorophore, respectively, and wherein each of        the first cell preparation and the second cell preparation        comprise the transcript of interest, and the adjacent codon pair        recognized by the first and second tRNA occurs in the codon        sequence of the transcript of interest at a frequency        significantly higher than the frequency of the translation        product of the same codon pair in the proteome of the cells;    -   (vi) introducing to the second cell preparation a drug        candidate;    -   (vii) measuring electromagnetic radiation emitted from the first        cell preparation; and    -   (viii) measuring electromagnetic radiation emitted from the        second cell preparation, wherein a significant difference        between the electromagnetic radiation measured in step (iii) and        the electromagnetic radiation measured in step (iv) indicates        that the drug candidate affects the translation of the        transcript of interest.

Step (iii) is performed under conditions wherein the first cellpreparation has not been contacted with the drug candidate. It will beunderstood to those of skill in the art that the cells can comprise thetranscript of interest by virtue either of the presence therein of DNAthat encodes the transcript of interest or by transfection of thetranscript of interest into the cells. In another embodiment, the secondcell preparation is substantially identical to the first cellpreparation, except that the second cell preparation has not beencontacted with the drug candidate. Each possibility represents aseparate embodiment of the present invention.

In another embodiment, the present invention provides a method formeasuring the effect of a drug candidate on translation of a transcriptof interest, the method comprising the steps of:

-   -   (v) introducing into each of a first preparation and a second        preparation comprising a subcellular compartment a first tRNA        and a second tRNA (i.e. a first tRNA pair), wherein the first        and second tRNA are labeled with a donor fluorophore and an        acceptor fluorophore, respectively, and wherein each of the        first and second preparations comprises the transcript of        interest, and the adjacent codon pair recognized by the first        and second tRNA occurs in the codon sequence of the transcript        of interest at a frequency significantly higher than the        frequency of the translation product of the same codon pair in        the proteome of the subcellular compartment;    -   (vi) introducing to the second preparation a drug candidate;    -   (vii) measuring electromagnetic radiation emitted from the first        preparation; and    -   (viii) measuring electromagnetic radiation emitted from the        second preparation, wherein a significant difference between the        electromagnetic radiation measured in step (iii) and the        electromagnetic radiation measured in step (iv) indicates that        the drug candidate affects the translation of a transcript of        interest.

Step (iii) is performed under conditions wherein the first preparationof the subcellular compartment has not been contacted with the drugcandidate. It will be understood to those of skill in the art that thesubcellular compartment can comprise the transcript of interest byvirtue either of the presence therein of DNA that encodes the transcriptof interest or by transfection of the transcript of interest into thesubcellular compartment. In another embodiment, the second preparationis substantially identical to the first preparation, except that thesecond preparation has not been contacted with the drug candidate. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, a method of the present invention furthercomprises the steps of introducing into the cell or cells being analyzeda second tRNA pair, i.e. a third tRNA labeled with a second donorfluorophore and a fourth tRNA labeled with a second acceptorfluorophore. The adjacent codon pair recognized by the second tRNA pairoccurs in the codon sequence of the transcript of interest at afrequency that is significantly lower than the frequency of thetranslation product of the same codon pair in the proteome of the cell.The labeled second tRNA pair produces a second FRET signal distinct fromthe FRET signal produced by the labeled first tRNA pair. In thisembodiment, the second FRET signal reflects general translation in thecell being assayed, not including translation of the transcript ofinterest. In another embodiment, translation of the transcript ofinterest makes a relatively small contribution to the second FRETsignal. In either case, analysis of the first and second FRET signalsenables accurate determination of the level of translation of thetranscript of interest. In another embodiment, the adjacent occurrenceof the first and second tRNA is significantly lower in the transcript ofinterest, relative to the transcriptome of the cell.

In another embodiment, a method of the present invention furthercomprises the steps of introducing into a cell or subcellularcompartment thereof under analysis, a second tRNA pair, i.e. a thirdtRNA labeled with a second donor fluorophore and a fourth tRNA labeledwith a second acceptor fluorophore, wherein the adjacent codon pairrecognized by the third tRNA and the fourth tRNA occurs in the codonsequence of the transcript of interest at a frequency significantlylower than in the proteome of the cell or subcellular compartment. Thesecond tRNA pair thus produces a second FRET signal distinct from theFRET signal produced by the first tRNA pair.

In another embodiment, a method of the present invention furthercomprises the steps of introducing into an additional cell or anadditional subcellular compartment a second tRNA pair, i.e. a third tRNAlabeled with a second donor fluorophore and a fourth tRNA labeled with asecond acceptor fluorophore, wherein the codons recognized by the thirdtRNA and the fourth tRNA occur adjacently in the codon sequence of thetranscript of interest at a frequency that is significantly lower thanthe frequency of the translation product of the same codon pair in theproteome of the cell or subcellular compartment.

In another embodiment, a method of the present invention furthercomprises the step of irradiating the system or biological sample with asource of electromagnetic radiation prior to the step of detecting theelectromagnetic radiation emitted in the form of FRET signals. Thissource produces electromagnetic radiation of a different wavelength thanthat detected as a readout of protein translation activity. In anotherembodiment, the wavelength of electromagnetic radiation produced by thissource is the excitation wavelength of a marker of the presentinvention. In another embodiment, the wavelength is the excitationwavelength of the donor fluorophore of the FRET pair contained in thefirst and second tRNA.

In another embodiment, a method of present invention is used to monitortranslation of a group of specific proteins. In this embodiment, amethod of the present invention is generalized to more than 2 labels,yielding a system of equations is be solved for the unknowns. In anotherembodiment, all 210 pairs are used, for example in a multi-well plate.The cells at hand are placed in the well plate and the tRNA pairs andrequired reagents are added. Once the FRET signals have been measured, acomputer program analyzes the 210 numbers and outputs the requiredresults. In another embodiment, this method is used to define a proteinproduction profile or “fingerprint,” which can then be used for purposessuch as quality assurance, cell characterization, and drug development.

According to one embodiment, the method of the present invention furthercomprises the step of analyzing the electromagnetic radiation or asignal produced therefrom, thereby obtaining a read-out in real-time ofthe translation of a transcript of interest

According to another embodiment, a method of the present inventionfurther comprises the step of computing the number of events (N) over aperiod of time t, wherein

$\left. N \right.\sim\frac{\Sigma \; I_{t}^{2}}{\Sigma \; \delta \; I_{t}^{2}}$

wherein I_(t) is the average signal strength at time t and δI_(t) is theaverage signal deviation at time t, thereby obtaining a readout oftranslation of the transcript of interest. “Average signal deviation”refers, in another embodiment, to the detected signal minus the averagesignal.

In another embodiment, a plurality of cells or subcellular compartmentscomprising the labeled element is utilized in a method of the presentinvention.

In one exemplary embodiment, the transcript of interest encodes insulin.In another embodiment, the transcript of interest encodes collagen orelastin. In another embodiment, the transcript of interest encodes agrowth factor. In another embodiment, the transcript of interest encodeserythropoietin. In another embodiment, the transcript of interestencodes a stem cell specialization factor. In another embodiment, thetranscript of interest encodes a protein selected from insulin, a growthfactor, erythropoietin, a stem cell specialization factor and aproteosome inhibitor. Each possibility represents a separate embodimentof the present invention.

In another embodiment, the protein of interest of the present inventionis an antibody. In another embodiment, the antibody is a therapeuticantibody. In another embodiment, the antibody is a diagnostic antibody.In another embodiment, the antibody has a known utility selected from atherapeutic utility and a diagnostic utility.

In another embodiment, the protein of interest is selected from thegroup consisting of insulin, a growth factor, an antibody,erythropoietin, and a stem cell specialization factor. In anotherembodiment, the transcript of interest encodes another recombinantprotein used for a therapeutic purpose. In another embodiment, thetranscript of interest encodes another recombinant protein used for adiagnostic purpose. Each possibility represents a separate embodiment ofthe present invention.

In another embodiment, the protein of interest is selected from thegroup consisting of CPF-DD, Peptide T, vasoactive intestinal peptide,tetradecapeptide, erythropoietin, insulin, growth hormone, pentigetide,histamine releasing peptide antigen, vasoactive intestinal peptideanalog, corticotropin releasing factor, corleukin, CY 725, CY 726,pancreatic trypsin inhibitor, somatostatin, calcium channel peptide,Ebiratide, DGAVP, E-2078, DPDPE, Dynorphin A, sleep inducing peptide,calcitonin, PTH-releasing peptide, growth hormone releasing peptide,HCG, hirudin, an hirudin analog, applagin, corplatin, integrelin, aviper venom polypeptide, desmopressin, antistasin, EGF receptor blocker,Bestatin, buserelin, goserelin, leuprolide, TGF beta, atrial natriureticpeptide, auriculin, brain neuritic peptide, urodilatin, captopril, ACEinhibitor peptide, and a renin inhibitor.

In another embodiment, the protein of interest of the present inventionis selected from the group consisting of growth hormone, prolactin,placental lactogen, luteinizing hormone, follicle-stimulating hormone,chorionic gonadotropin, thyroid-stimulating hormone, leptin,adrenocorticotropin, angiotensin I, angiotensin II, beta-endorphin,beta-melanocyte stimulating hormone, cholecystokinin, endothelin I,galanin, gastric inhibitory peptide, glucagon, insulin, lipotropins,neurophysins, somatostatin, calcitonin, calcitonin gene related peptide,beta-calcitonin gene related peptide, hypercalcemia of malignancyfactor, parathyroid hormone-related protein, parathyroid hormone-relatedprotein, glucagon-like peptide, pancreastatin, pancreatic peptide,peptide YY, PHM, secretin, vasoactive intestinal peptide, oxytocin,vasopressin, vasotocin, enkephalinamide, metorphinamide, alphamelanocyte stimulating hormone, atrial natriuretic factor, amylin,amyloid P component, corticotropin releasing hormone, growth hormonereleasing factor, luteinizing hormone-releasing hormone, neuropeptide Y,substance K, substance P, and thyrotropin releasing hormone.

In another embodiment, the protein of interest of the present inventionis selected from the group consisting of carbamoyl synthetase I,ornithine transcarbamylase, arginosuccinate synthetase, arginosuccinatelyase, arginase, fumarylacetoacetate hydrolase, phenylalaninehydroxylase, alpha-1 antitrypsin, glucose-6-phosphatase,low-density-lipoprotein receptor, porphobilinogen deaminase, factorVIII, factor IX, cystathione beta-synthase, branched chain ketoaciddecarboxylase, albumin, isovaleryl-CoA dehydrogenase, propionyl CoAcarboxylase, methyl malonyl CoA mutase, glutaryl CoA dehydrogenase,insulin, beta-glucosidase, pyruvate carboxylase, hepatic phosphorylase,phosphorylase kinase, glycine decarboxylase, H-protein, T-protein,Menkes disease copper-transporting ATPase, Wilson's diseasecopper-transporting ATPase, cytosine deaminase, hypoxanthine-guaninephosphoribosyltransferase, galactose-1-phosphate uridyltransferase,phenylalanine hydroxylase, glucocerbrosidase, sphingomyelinase,beta-L-iduronidase, glucose-6-phosphate dehydrogenase,glucosyltransferase, HSV thymidine kinase, and human thymidine kinase.

According to some embodiments, the protein of interest further comprisesa protein tag. According to other embodiments, the protein of interestis a fusion protein comprising a protein tag. According to otherembodiments, the protein tag is selected from the group consisting of anaffinity tag, a solubilization tag, a chromatography tag, an epitope tagand a fluorescent protein tag. It is to be specifically understood thata single protein tag may fall into more than one of the aforementionedcategories.

Examples of protein tags include, without limitation, protein A;biotin-carboxy carrier protein (BCCP; SEQ ID NO:1) tag; c-myc tag(EQKLISEEDL; SEQ ID NO:2); calmodulin tag; FLAG-tag (DYKDDDDK; SEQ IDNO:3); IRS (RYIRS; SEQ ID NO:4); AU1 (DTYRYI; SEQ ID NO:5); AU5 (TDFLYK;SEQ ID NO:6); hemagglutinin (HA) tag; KT3 (KPPTPPPEPET; SEQ ID NO:7);VSV-G (YTDIEMNRLGK; SEQ ID NO:8); polyhistidine (His-tag); maltosebinding protein tag; Nus tag; glutathione-S-transferase (GST) tag; greenfluorescent protein (GFP) tag; thioredoxin; S-tag; Strep-tag (WSHPQFEK;SEQ ID NO:9); and T7 tag.

In another embodiment, the present invention provides a method ofperforming quality assurance of expansion of a stem cell population ofinterest, wherein the stem cell population expresses a stem cellspecialization factor, the method comprising the steps of (i)introducing into the stem cell population a first tRNA and second tRNA,wherein the first tRNA is labeled with a donor fluorophore, the secondtRNA is labeled with an acceptor fluorophore, and the adjacent codonpair recognized by the first tRNA and the second tRNA occurs in thecodon sequence of a transcript encoding the stem cell specializationfactor at a frequency that is significantly higher than the frequency ofthe translation product of the same codon pair in the proteome of thecell population; and (ii) detecting electromagnetic radiation emittedfrom the stem cell population. In another embodiment, the adjacent codonpair is enriched in the transcript encoding the stem cell specializationfactor, relative to the transcriptome of the cell population.

In another embodiment, the present invention provides a method ofperforming quality assurance of production of a recombinant protein ofinterest in a cell population, the method comprising the steps of

-   -   (iii) introducing into the population a tRNA pair comprising a        first tRNA and second tRNA, wherein one of the first tRNA or the        second tRNA is labeled with a donor fluorophore, and the other        is labeled with an acceptor fluorophore, wherein the donor        fluorophore and the acceptor fluorophore together form a FRET        pair; and wherein the adjacent codon pair recognized by the        first tRNA and the second tRNA occurs in the codon sequence of a        transcript encoding the recombinant protein at a frequency that        is significantly higher than the frequency of the translation        product of the same codon pair the proteome of the cell        population; and    -   (iv) detecting FRET signals emitted from the cell population. In        another embodiment, the adjacent occurrence of the first and        second tRNA is enriched in the transcript encoding the        recombinant protein, relative to the transcriptome of the cell        population.

In another embodiment, the recombinant protein is a fusion proteincomprising a protein of interest and a protein tag. In a particularembodiment, the adjacent codon pair recognized by the first tRNA and thesecond tRNA occurs in the codon sequence of the protein tag. In aparticular embodiment, the adjacent codon pair recognized by the firsttRNA and the second tRNA occurs in the codon sequence of the mRNAencoding the protein of interest.

In a particular embodiment, the recombinant protein of interest is arecombinant antibody. In another embodiment, the cell population ofcomprises a recombinant antibody-producing cell population.

In another embodiment, the above method is used to facilitate cloneenrichment in the process of isolation of a clone producing a desiredamount of the target antibody.

In another embodiment, the above method is used to facilitate clonesorting in the process of isolation of a clone producing a desiredamount of the target antibody.

In another embodiment, the present invention provides a method ofperforming quality assurance of antibody production in a population of arecombinant antibody-producing cell type.

In another embodiment, the antibody of the above methods is atherapeutic antibody. In another embodiment, the antibody is adiagnostic antibody. In another embodiment, the antibody has a knownutility selected from a therapeutic utility and a diagnostic utility.

In another embodiment, the present invention provides a multi-wellcontainer comprising 2 or more wells, each of which is labeled with aspecific pair of labeled tRNA sets. “tRNA set,” in this embodiment,refers to all tRNA associated with a particular amino acid in a cell tobe tested. In another embodiment, the present invention provides amulti-well container comprising 3 or more wells, each of which islabeled with a pair of labeled tRNA sets. In another embodiment, thepresent invention provides a multi-well container comprising 5 or morewells, each of which is labeled with a pair of labeled tRNA sets.

In another embodiment, the present invention provides a multi-wellcontainer comprising 10 or more wells, each of which is labeled with apair of labeled tRNA sets. In another embodiment, the present inventionprovides a multi-well container comprising 20 or more wells, each ofwhich is labeled with a pair of labeled tRNA sets. In anotherembodiment, the present invention provides a multi-well containercomprising 50 or more wells, each of which is labeled with a pair oflabeled tRNA sets. In another embodiment, the present invention providesa multi-well container comprising 100 or more wells, each of which islabeled with a pair of labeled tRNA sets. In another embodiment, thepresent invention provides a multi-well container comprising 150 or morewells, each of which is labeled with a pair of labeled tRNA sets. Inanother embodiment, the present invention provides a multi-wellcontainer comprising 200 or more wells, each of which is labeled with apair of labeled tRNA sets. In another embodiment, the present inventionprovides a multi-well container comprising 210 wells, each of which islabeled with a pair of labeled tRNA sets.

In another embodiment, the present invention provides the use of amulti-well container of the present invention to measure translation ofa protein of interest. In another embodiment, the present inventionprovides use of a multi-well container of the present invention tocharacterize the translational profile of a cell of interest. In anotherembodiment, the multi-well container is designed for cells to becharacterized and transfection agents to be introduced to each well,followed by assessment of translation according to a method of thepresent invention. Each possibility represents a separate embodiment ofthe present invention.

In another embodiment, the present invention provides a multi-wellcontainer comprising 2 or more wells, each of which is labeled with apair of labeled tRNAs. In another embodiment, the present inventionprovides a multi-well container labeled with 3 or more pairs of labeledtRNAs. In another embodiment, the present invention provides amulti-well container labeled with 5 or more pairs of labeled tRNAs. Inanother embodiment, the present invention provides a multi-wellcontainer labeled with 10 or more pairs of labeled tRNAs. In anotherembodiment, the present invention provides a multi-well containerlabeled with 20 or more pairs of labeled tRNAs. In another embodiment,the present invention provides a multi-well container labeled with 50 ormore pairs of labeled tRNAs. In another embodiment, the presentinvention provides a multi-well container labeled with 100 or more pairsof labeled tRNAs. In another embodiment, the present invention providesa multi-well container labeled with 150 or more pairs of labeled tRNAs.In another embodiment, the present invention provides a multi-wellcontainer labeled with 200 or more pairs of labeled tRNAs. In anotherembodiment, the present invention provides a multi-well containerlabeled with 300 or more pairs of labeled tRNAs. In another embodiment,the present invention provides a multi-well container labeled with 400or more pairs of labeled tRNAs. In another embodiment, the presentinvention provides a multi-well container labeled with 500 or more pairsof labeled tRNAs. In another embodiment, the present invention providesa multi-well container labeled with 600 or more pairs of labeled tRNAs.In another embodiment, the present invention provides a multi-wellcontainer labeled with 700 or more pairs of labeled tRNAs. In anotherembodiment, the present invention provides a multi-well containerlabeled with 800 or more pairs of labeled tRNAs. In another embodiment,the present invention provides a multi-well container labeled with 900or more pairs of labeled tRNAs. In another embodiment, the presentinvention provides a multi-well container labeled with all 990 pairs oflabeled tRNAs. In another embodiment, the present invention provides theuse of a multi-well container of the present invention to measuretranslation of a protein of interest. In another embodiment, the presentinvention provides use of a multi-well container of the presentinvention to characterize the translational profile of a cell ofinterest. In another embodiment, the multi-well container is designedfor cells to be characterized and transfection agents to be introducedto each well, followed by assessment of translation according to amethod of the present invention. Each possibility represents a separateembodiment of the present invention.

In another embodiment, once electromagnetic radiation of the requiredwavelength and energy has been administered to the biological sample,thereby exciting the donor fluorophores, an optical apparatus monitorsfluorescence emanating from the cellular translation system. Theacceptor fluorophores on the tRNA respond to this energy with the FRETsignal whenever a donor and acceptor pair are in sufficient proximity,indicative of particular steps of translation activity. Fluorescentradiation emitted from acceptor fluorophores is detected by the opticalapparatus and the event is recorded by an image acquisition device.

In another embodiment, an acceptor fluorophore of the present inventionemits a signal that is detectable through a means of detectingelectromagnetic radiation “Detectable” as used herein refers to a signalable to be detected, over the background level, by standard means ofdetecting electromagnetic radiation. Means of detecting electromagneticradiation are well known in the art. In some preferred embodiments, thesignal is detected using total internal reflection fluorescencemicroscopy (TIR-FM) “TIR-FM” as used herein refers to a microscopyillumination method that illuminates a very small volume at theinterface of two materials with different refractive indices. TIR-FM isdescribed in WO 05/116252 and in US patent applications 2004/0023256 and2006/0228708, which are incorporated herein in their entirety byreference.

Another microscopy method uses Low Angle Oblique (LAO) illumination fordetecting optical signals deeper within cells, as described in Sako Y,Yanagida T (2003) Single-molecule visualization in cell biology. Nat RevMol Cell Biol 4 (Suppl): SS1-SS5

Additional means of detecting electromagnetic radiation include imageacquisition devices; confocal laser scanning microscopes (LSM), used toimprove fluorescence image quality by eliminating out-of-focusfluorescence; and spinning disk confocal microscopes, which can includevideo rate (typically 30 frames per second) imaging with charge-coupleddevice (CCD) cameras and imaging of 3-dimensional structures in livingcells on a sub-second time scale with reducedphotobleaching/phototoxicity (Graf et al, Live cell spinning diskmicroscopy. Adv. Biochem. Eng. Biotechnol. 95: 57-75, 2005).Programmable array microscopes (Hanley et al, An optical sectioningprogrammable array microscope implemented with a digital micromirrordevice, J Microsc 196: 317-331, 1999) and line scanning microscopes areavailable and offer similar advantages to spinning-disk confocals. Inaddition, multi-photon microscopes use infrared light, which readilypenetrates up to 600 μm, allowing deep tissue imaging in living animals(Helmchen and Denk, Deep tissue two-photon microscopy. Nat Methods 2:932-940, 2005). Additional methods are described inter alia in WO2007/002758, WO 2008/028298, European Patent EP1428016, and U.S. Pat.No. 7,015,486 and US Patent application 2005/0157294, which areincorporated herein by reference. Each method represents a separateembodiment of the present invention.

According to one embodiment, the translation apparatus is placed in atest-tube and manually observed. In another embodiment, the system isplaced in a multi-well plate such as a 96 or 384 well plate and observedby a high-throughput fluorimetry instrument.

According to another embodiment, the translation apparatus is placedunder a microscope suitable for observing fluorescence at subcellularresolution, such as instruments available from Zeiss (Oberkochen,Germany) and Leica (Wetzlar, Germany), with an image acquisition deviceoperable at a sufficient rate (10-100 frames per second) andcomputational units that can acquire and analyze the resulting imagesand data.

Assessing cellular translation activity can be accomplished in a varietyof ways according to methods of the present invention. In oneembodiment, a well of a 96-well plate or other commercially availablemulti-well plate is used to contain the biological sample. In anotherembodiment, the receptacle is the reaction vessel of a FACS machine.Other receptacles useful in the present invention include, but are notlimited to 384-well plates. Still other receptacles useful in thepresent invention will be apparent to the skilled artisan to facilitaterapid high-throughput screening.

Overview of One Exemplary Embodiment of the Present Invention

A tRNA or a plurality thereof is engineered to carry a donor fluorophoreand utilized as a donor, and a different tRNA or a plurality thereof isengineered to carry an acceptor fluorophore and utilized as an acceptor.The labeled tRNA are introduced into cultured cells or subcellularcompartments. In order to monitor translation, a light sourceilluminates the cells, thus exciting the donor fluorophores and therebythe acceptor fluorophores whenever these components are in sufficientproximity to each other, generating a measurable signal.

If the labeled FRET pair, during the process of translation, is broughtinto close proximity, (often this distance is around 5 nm), a FRETsignal is observed. When they are separated, the signal ceases. Sincethe distance in the ribosomes between A-Site and P-Site is about 35Angstroms, and between the P-Site and E-Site is between 17-60 Angstroms(see Agrawal et al, Visualization of tRNA Movements on the Escherichiacoli 70s Ribosome during the Elongation Cycle, J. Cell Biol. 2000 August7′ 150(3):447-460) and since the duration time of tRNA immobilization inthe eukaryotic ribosome is around 0.5 second, a strong FRET signal isgenerated. Thus, generation of FRET signals from this pair indicatestranslation activity. The measurement can be the intensity of the signalor any other relevant feature, such as signal variability, signalpolarity, signal lifetime, wavelength, photon number, spectrum, etc. aswill be appreciated by one skilled in the art of fluorescent labelingand measurements.

One exemplary measurement measures the variability of the emittedsignal. From this variability, it is possible to deduce the number ofon/off events in the sample being measured. This is similar tomeasurements performed in Fluorescent Correlation Spectroscopy (FCS). Inthese applications, signal variation is measured and used for computingbasic parameters of the system, such as the number of fluorescingmolecules in the system. In FCS, the variability is mainly a function ofmolecules entering and leaving the illuminated volume. In an exemplaryembodiment of the present invention, the variation is mainly caused by“blinking” (turning on and off) of the signals in response to proteintranslation activity. Thus, the translation activity being detected isevaluated from the ratio of variation to average signal intensity.Consequently, a signal that does not vary, e.g., in the event that thesignal is constantly ON, is interpreted as lack of translation activity.

To compute the number of events, a person skilled in the art can use anysuitable method known in the art, including, but not limited to, amethod where the signal is measured over a period of time (preferablymeasured in seconds), and the autocorrelation is computed as follows:

$\left. N \right.\sim\frac{\Sigma \; I_{t}^{2}}{\Sigma \; \delta \; I_{t}^{2}}$

where I_(t) is the signal strength at time t, and δI_(n) is the signaldeviation at time t (signal−average signal). In this way of measuringsignal variations (with the accepted assumption that blinking follows aPoisson/Gaussian distribution), an estimate can be obtained on the eventrate in the observed volume.

Introduction of tRNA and Nucleic Acid Molecules into a Target Cell

According to one embodiment of the present invention, labeled tRNAs areintroduced into intact cells. This can be accomplished through a varietyof established methods such as encapsulation of tRNA into liposomes orvesicles which are capable of fusion with cells. Fusion introduces theliposome or vesicle interior solution containing the tRNA into the cell.Alternatively, some cells will actively incorporate liposomes into theirinterior cytoplasm through endocytosis. The labeled tRNAs can also beintroduced through the process of cationic detergent mediatedlipofection (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413-17,1987), or injected into large cells such as oocytes.

Additional methods for introduction of tRNA into a target cell are wellknown in the art. Such methods include the use of RNAiFect™ from Qiagenof Valencia, Calif. (Sako et al ibid) and electroporation. Sako et aldiscloses transfection of tRNA molecules, engineered to carry ananticodon for one of the natural stop codons (CUA, UUA, UCA) into A549cells using the transfection agent RNAiFect (Qiagen, Hilden, Germany) isshown. The engineered tRNA were properly transfected and provedfunctional in a luciferase assay, where the luciferase gene includedstop codons UGA, UAA, or UAG in place of the native Ser170 codon.

In another embodiment, INTERFERin™ (Autogen Bioclear™, Wiltshire, UK) isused for tRNA transfection. INTERFERin™ has been successfully used fortRNA transfection

Additional methods for the introduction of nucleic acid molecules aredescribed in Akhtar et al., (Trends Cell Bio. 2, 139, 1992). WO 94/02595describes general methods for introduction of enzymatic RNA molecules.These protocols can be utilized for the introduction of virtually anynucleic acid molecule. Nucleic acid molecules can be administered tocells by a variety of methods known to those familiar to the art,including, but not restricted to, encapsulation in liposomes(WO03057164, Malone, R. W. et al., 1989, Proc. Natl. Acad. Sci. USA. 86:6077-6081; Glenn, J. S. et al., 1993, Methods Enzymol. 221: 327-339; Lu,D. et al., 1994, Cancer Gene Ther. 1: 245-252), by microinjection (Liuet al., 2005, Dev Growth Differ. 47(5):323-31), by iontophoresis(Sakamoto et al., 2004, Gene Ther. 11(3):317-24), or by incorporationinto other vehicles, such as hydrogels, cyclodextrins, biodegradablenanocapsules, and bioadhesive microspheres.

U.S. Patent Application No. 2004/235175 discloses a method of insertingRNA into cells. In this method, cells are transfected with RNA usingelectroporation in order to achieve high transfection efficiency.

In another, non-limiting exemplary electroporation protocol, 3-40×10⁶cells, preferably growing at log phase, are harvested, counted andwashed with cold 1× HeBS (Hepes-buffered saline). Cells are resuspendedin 0.8 mL 1× HeBS containing the tRNA and incubated at room temperaturefor 15 minutes. An exemplary recipe for HeBS is 20 mM HEPES, 150 mMNaCl, pH 7.0-7.4. The tRNA/cell suspension is transferred to anelectroporation cuvette and electroporated at an appropriate voltage,preferably at between 500-2000 μF capacitance. The time constant isrecorded if desired, and the mixture is optionally incubated in thecuvette for about 10 minutes at room temperature, prior to returning thecells to culture media.

In another, non-limiting exemplary electroporation protocol successfullyused for CHO-K1 cells, HEK cells, and rat hippocampal neurons (thushaving utility for a large variety of cell types), tRNA is precipitated(either alone or as a coprecipitate with DNA) in ethanol and ammoniumacetate at −20° C. for at least 1 hour. The precipitated tRNA ispelleted, vacuum dried, and resuspended in CO₂-independent medium to thedesired final concentration (4 μg/μl tRNA, either with our without 2.5μg/n1 carrier DNA, is typically appropriate). Immediately prior toelectroporation, the media is replaced with CO₂-independent media,containing no glutamine, FBS or antibiotics. CO₂-independent media areavailable e.g. from Invitrogen-Gibco and include phenol red free media,Liebovitz's L15 Media (catalogue no. 11415-114), and catalogue nos.18055-088; 18045-088, and 041-95180M. Approximately 5 μl ofelectroporation solution is added to the cells, followed by electricalpulse application. For CHO-K1 cells and HEK cells, four 120 V pulses of50 ms duration are typically used, and for neurons, four 160 V pulses of25 ms duration. The CO₂-independent media is immediately replaced withfresh Ham's F12 media for CHOK1 cells, DMEM for HEK cells, or neurobasalmedia for neurons, and cells are returned to the 37° C. incubator.

In another, non-limiting exemplary electroporation protocol,electrolyte-filled fused silica capillaries (30 cm long, 30-nm id.,375-nm od) are used. The outlet end of the capillaries is tapered to anapproximate outer tip diameter (typically 50 μm, depending on the sizeof the cell type used). Exemplary electrolytes useful in this method arethose based on HEPES buffer. The tapered outlet end of the capillary issubmerged in the buffer contained in the cell chamber, and the inlet endis placed in a buffer-filled vial. Both the capillary and the inlet vialbuffer solutions contain the tRNA and/or any other components to betransfected. Cells are placed in a chamber on the microscope stage, andcell bathing medium (HEPES buffer) is electrically grounded. Thecapillary outlet is placed within 5 nm of the cell surface, and the DChigh voltage power supply is connected.

In another, non-limiting exemplary electroporation protocol, cells areelectroporated using a modified patch-clamp technique. Single cellsunder direct observation are indented with a microelectrode andelectroporated using a current delivered from a simple voltage-clampcircuit, as described in detail in Rae J L and Levis R A, Single-cellelectroporation, Pflugers Arch 443(4):664-70, 2002.

In another, non-limiting exemplary electroporation protocol successfullyused for electroporation of DNA, but equally useful for tRNA, intoindividual neurons in cultures of organotypic brain slices (FIG. 3),micropipettes with a tip diameter of about 1-2 μm and with resistancesof 10-20 MΩ are pulled from capillary glass with filament (availablefrom Science Products, Hofheim, Germany, catalogue number GB150E-8P) ona Micropipette Puller (available from Sutter Instrument Company, Novato,USA catalogue number P-97). Micropipettes are mounted on a three-axismicromanipulator (Luigs and Neumann, Ratingen, Germany). A Millicell CMinsert (Millipore, Billerica, Mass., USA) containing a brain slice isplaced in a perfusion chamber on the stage of a Zeiss Axioplan™microscope and continuously perfused with oxygenated physiological saltsolution during electroporation. The overall time under perfusion istypically about 30 min. Slices are transferred back into the incubator,individual cell somata are identified, and a pipette tip is gentlyplaced against the cell membrane. Voltage pulses are delivered betweenan electrode placed inside the micropipette in contact with the tRNAsolution (cathode), and a ground electrode (anode) using an isolatedvoltage stimulator (available from WPI, Berlin, Germany, under the nameHI-MED HG-203) controlled by a tetanizer (available from SigmannElektronik, Hueffenbart, Germany). To prevent the tip from clogging anddilution of the tRNA, a back-pressure (typically 2-10 mbar) is appliedto the pipette. In an exemplary embodiment, a single train of 200 squarepulses with a duration of 1 ms is applied, using a 4 ms delay with anamplitude of 10 V. The 1 ms pulses remove the negatively charged tRNAfrom the pipette by electrophoresis, driving electroporation. Typically,no voltage is applied during the delay of 4 ms between the pulses andthus there is no current flowing through the circuit.

Each method for introduction of tRNA into a cell represents a separateembodiment of the present invention.

Introduction of tRNA into Subcellular Compartments

Vestweber and Schatz (Nature 338: 170-172, 1989) achieved uptake of bothsingle- and double-stranded oligonucleotides into yeast mitochondria bycoupling the 5′ end of the oligonucleotide to a precursor proteinconsisting of the yeast cytochrome c oxidase subunit. Seibel et al.(Nucleic Acids Research 23: 10-17, 1995) have described the import intothe mitochondrial matrix of double-stranded DNA molecules conjugated tothe amino-terminal leader peptide of the rat ornithine-transcarbamylase.

Methods for the introduction of nucleic acid molecules into the interiorof an organelle are disclosed in WO2003/052067. WO2005/001062 disclosesthe use of viral vectors that contain localization signals specific forthe target organelle. These protocols can be utilized for theintroduction of labeled tRNAs into a mitochondria or chloroplast.

Labeling and Detection According to the Present Invention

In other embodiments, methods of the invention can be carried out inaccordance with the following alternatives:

tRNA Labeling.

Methods for fluorophore labeling of tRNA (FIG. 1) are well known in theart and are described inter alia in U.S. Pat. No. 7,288,372 and U.S.Patent applications 2003/0219780 and 2003/0092031, which areincorporated herein by reference.

In another exemplary method, used for Met-tRNA (Jun S Y et al,Fluorescent labeling of cell-free synthesized proteins withfluorophore-conjugated methionylated tRNA derived from in vitrotranscribed tRNA. J Microbiol Methods. 2008 June; 73(3):247-51) butsuitable for any tRNA, 10 μl of 30 mM succinimidyl ester of fluorescentdye in dimethy sulfoxide (DMSO) is added to 40 μl of theMet-tRNAfMet-resuspended solution and incubated for 40 min on ice. Thereaction is stopped by adding one-tenth volume of 2M sodium acetate, pH5.0. Fluorophore-conjugated Met-tRNAfMet is extracted repeatedly with anequal volume of acid phenol:chloroform (1:1, v/v; pH 5.0. Two and a halfvolumes of cold 95% (v/v) ethanol solution are added to the aqueousphase, and the mixture is allowed to stand at −70° C. for 1 h toprecipitate fluorophore-conjugated Met-tRNAfMet. The precipitated pelletis collected by micro-centrifugation at 14,000 rpm at 4° C. for 20 min,and then resuspended in an equal volume of diethyl pyrocarbonate(DEPC)-treated water to the original reaction volume. After alcoholprecipitation, the precipitate is washed with 80% (v/v) ethanolsolution, dried under vacuum, and resuspended in 20 μl of DEPC-treatedwater.

In another exemplary method, used for conjugation of BODIPY-FL toMet-tRNA (Olejnik J et al, N-terminal labeling of proteins usinginitiator tRNA. Methods. 2005 July; 36(3):252-60), but suitable forconjugation of BODIPY-FL to any tRNA, 1.0 OD260 (1500 pmol) ofmethionyl-tRNAfMet (tRNAfMet [Sigma Chemicals, St. Louis, Mo.],aminoacylated with methionine) is dissolved in water (37.5 μl), followedby addition of 2.5 μl of 1N NaHCO₃ (final conc. 50 mM, pH 8.5), followedby 10 μl of 10 mM BODIPYFL-SSE solution (Molecular Probes, Eugene,Oreg.). The modification reaction is allowed to proceed for 10 min at 0°C. and quenched by the addition of 0.1 volume of 1M lysine. 0.1 volumeof 3M NaOAc, pH 5.0, is added, and modified tRNA is precipitated with 3volumes of ethanol, dissolved in 50 μl of water, and purified on a NAP-5column (Amersham-Pharmacia, Piscataway, N.J.) to remove any freefluorescent reagent.

In general, tRNA molecules can be tagged while retaining theirinteraction with the aminoacyl synthetases as well as retaining theirfunctionality with the ribosome. tRNAs have been tagged with fluorescein(Watson et al., 1995, Biochemistry. 34 (24): 7904-12), with tetra methylrhodamine (TMR) (Jia et al., 1997, Proc Natl Acad Sci USA. 7932-6), andwith proflavine and ethidium bromide (Wintermeyer and Zachau, 1971, FEBSLett. 18 (2): 214-218). In another embodiment, the fluorophore isselected from the group consisting of fluorescein, rhodamine, proflavineand ethidium bromide.

Certain preferred embodiments of the present invention include labelingthe tRNA with small organic dyes attached to the “shoulder” region ofthe tRNA, such as in positions 8 and 47 of E. coli tRNAs, which havebeen often used for this purpose. One particular labeling method isattaching the label of choice to one or both of the dihydrouridines inthe D-Loop of the tRNA. Most tRNA have these dihydrouridinemodifications, enabling a wide choice of labels, including rhodamines,which are very useful due to their low tendency to bleach and highsignal strength. The most widely used dyes are FITC and (excitation andemission peaks at 490 nm and 520 nm) and TMR (excitation peaks at 550 nmand emission at 573 nm).

Typically, FRET occurs only when neighboring sites in the ribosome (forexample A and P, or P and E) are occupied by a donor-acceptor pair. Forexample, if 10% of all cellular tRNA is labeled, then on averageapproximately 1% of active ribosomes will be in a FRET configuration(0.25% in each of PA, AP, PE, EP configurations, wherein A,P,E indicatethe ribosomal tRNA sites, and donor is in the first and acceptor in thesecond site).

According to another embodiment of the present invention, the ratio ofimmobilized tRNAs in adjacent ribosomal sites is detected by measurementof FRET resulting from interaction between donor and acceptorfluorophores attached to the corresponding tRNAs. When both the donorand the acceptor fluorophores are attached to one or more species of thetRNAs, an elongation activity is detected.

In certain preferred embodiments, fluorophores utilized in the presentinvention exhibit a high quantum yield of fluorescence at a wavelengthdifferent from native cell components; e.g. nucleic acids and aminoacids. Upon excitation at a pre-selected wavelength, the marker isdetectable at low concentrations either visually or using conventionalfluorescence detection methods.

Systems and Methods for Study of Translation in Subcellular Compartmentsand Uses Thereof.

Methods of the present invention enable monitoring of translation ofselected transcripts of interest in various specific subcellularcompartments such as mitochondria, chloroplasts, and dendritic spines.In mitochondria and chloroplasts, the entire translation apparatus,including ribosomes, ribosomal proteins, translation factors, tRNAs andthe genetic code, are specific to the subcellular compartment anddistinct from those of the host eukaryotic cell. Also, apart from theribosomal RNA and tRNA, other proteins of the translation apparatus aresynthesized in the cell cytoplasm and imported into the subcellularcompartment. This allows a specific assay to be developed, wherein thetRNA of choice are labeled in the cell. In another embodiment, the tRNAare labeled in an isolated subcellular compartment. In both cases, thelabeled tRNA are directed to and imported into the subcellularcompartment. Thus the measured signals pertain to subcellularcompartment only and not to the general cellular translation apparatus.

Host Cells

Any cell is suitable for assaying translation by methods of the presentinvention. Non-limiting examples of target cell types are COS, HEK-293,BHK, CHO, TM4, CVI, VERO-76, HELA, MDCK, BRL 3A, NIH/3T3 cells, etc.Additional cell lines are well known to those of ordinary skill in theart, and a wide variety of suitable cell lines are available from theAmerican Type Culture Collection, 10801 University Boulevard, Manassas,Va. 20110-2209. Cells of particular interest include neuronal cells,immune system cells, including lymphocytes (B and T cells e.g., T helpercells) and leucocytes (e.g., granulocytes, lymphocytes, macrophage andmonocytes), cells from lymph, spleen and bone marrow tissues, epithelialcells, and cells from or derived from internal organs.

Signal Detection

The signals emitted by the cells or organelles of the present inventioncan be detected by a variety of different instrument configurations. Asa bulk assay, they can potentially be read manually, by comparing thefluorescent signal to calibrated standards under a fluorescent reader.Alternatively, they can be read by a fluorescent plate reader, made for96-well plates, 384-well plates, or another configuration known in theart. In another embodiment, the labeled cells are imaged by amicroscope, to identify subcellular localization of protein synthesisprocesses and to estimate the relative rates of protein synthesis invarious regions of the cell. In further embodiments, instruments capableof single-molecule detection in living cells are used.

Signal Analysis

There are numerous methods to process and analyze the resulting signals.In one embodiment, donor, acceptor and FRET signals are separatelymeasured and compared to yield the fraction of pairs in FRET positionversus the total concentration of donors and acceptors separately. Whenrepeating such measurements with various concentrations of labeledcomponents vs. unlabeled samples, it is possible to derive the overallfraction of component pairs versus the total number of components. Forexample, if 10% of tRNA are labeled as donors and 10% as acceptors, thenabout 4% of ribosomes will include a FRET tRNA pair in neighboringribosomal positions (1% AP, 1% PA, 1% PE, 1% EP, where donor is alwaysfirst and acceptor second). This creates a specific ratio ofdonor/acceptor/FRET signals. If only 5% are labeled as donors oracceptors, than only 1% FRET signal will occur. Thus the FRET signalstrength is proportional to the square of the donor/acceptor signals.This allows a calibration curve to be derived, for example in cell-freesystem, and later used in living cells to provide a precise estimate ofthe relative concentration of components in FRET position as well as theconcentration of all components.

In another embodiment, signal variance is computed, and the square ofthe ratio of average signal to average variance is computed, whichyields an estimation of the number of labeled components being measured.This assumes that the process underlying this variation is of Poissonianor Gaussian nature, such as in molecules diffusing into and out of acertain volume, or the blinking of labeled ribosomes in response toprotein synthesis. When considering a sizable number of ribosomes (10 ormore), the process can be assumed to be governed by Poissonian orGaussian statistics, depending on the number. In such cases, as is wellknown, the variability of the signal is proportional to the square rootof the signal strength. For example, let the measured signal be denotedby St, and let its average over a period of time (for example a fewseconds) be denoted by S_(av). The varianceVar(S)=average(S_(t)−S_(av)). In such processes, the size of thevariance is on average the square root of the signal. ThusVar(S)−sqrt(S_(av))=sqrt(NS) where S is the signal from a single event(for example FRET from a pair of labeled components). This means that(S/Var(S))²=NS/S=N or the number of active particles.

Application of the Present Invention for Diagnostic Applications

The methods disclosed herein are suitable for diagnostic applications,wherein rates of protein synthesis are indicative of type or phase of adisease or condition. For the purpose of diagnosis, cells are obtainedfrom the host, for example, from biopsy, and prepared for the assay. Inanother embodiment, the preparation comprises the following steps:

(a) introducing the labeled tRNA into the cells by means oftransfection; and

(b) detecting radiation emitted from the cells.

In another embodiment, the method further comprises the step ofanalyzing the radiation or a signal derived thereof, thereby obtaining areadout of translation activity.

Prior to detection, cells are commonly transferred to a carrier. Thetype of carrier depends on the type of measurement that is used fordetection. Thus, a carrier includes, but is not limited to, afluorescent plate reader.

In another embodiment, the above method is applied in a high-throughputoperation. In another embodiment, the method is applicable for accuratemeasurements of subcellular localization of protein synthesis events,for example, detection of translation activity in mitochondria orneuronal spines.

According to some embodiments, the method further comprises comparingthe amount of detected radiation to a reference standard, wherein alevel of detected radiation different from the reference standard isindicative of a particular disease or disorder. In another embodiment,the level of detected radiation is diagnostic for a disease, disorder orpathological condition. In other embodiments, the readout provided uponanalysis of the detected electromagnetic radiation is indicative of ordiagnostic for a disease, disorder or pathological condition.

Pathological conditions that are amenable to diagnosis using methods ofthe invention include without limitation, fragile X mental retardation,autism, aging and memory degeneration.

Diseases that are amenable to diagnosis using methods of the inventioninclude without limitation, mitochondria-related disease, cardiachypertrophy, restenosis, diabetes, obesity, a genetic disease resultingfrom a premature termination codon (PTC), and inflammatory boweldisease.

Other conditions that are amenable to diagnosis using methods of theinvention include malignant and pre-malignant conditions. Malignant andpre-malignant conditions include those which occur in hematologicalcells, such as a hematological malignancy. Hematological malignanciesinclude without limitation, acute lymphoblastic leukemia (ALL); acutemyelogenous leukemia (AML); chronic myelogenous leukemia (CML);Hodgkin's disease; non-Hodgkin lymphoma; chronic lymphocytic leukemia(CLL); diffuse large B-cell lymphoma (DLBCL); follicular lymphoma (FL);mantle cell lymphoma (MCL); hairy cell leukemia (HCL); marginal zonelymphoma (MZL); Burkitt's lymphoma (BL); post-transplantlymphoproliferative disorder (PTLD); T-cell prolymphocytic leukemia(T-PLL); B-cell prolymphocytic leukemia (B-PLL); Waldenstrom'smacroglobulinemia and multiple myeloma (MM). In one embodiment, themalignant disorder is multiple myeloma.

Pre-malignant conditions include without limitation, monoclonalgammopathy of uncertain significance and smoldering multiple myeloma.

Application of the Present Invention for High-Throughput Screening (HTS)Assays

The methods disclosed herein can optionally be used for the screening ofa large library of small molecules, recombinant proteins, peptides,antibodies, or other compounds to determine their efficacy or theirpotential for use as drugs, based on measuring the effect of a testcompound on translation of a specified protein in a test cell.High-throughput screening utilizes an assay that is compatible with thescreening instrument, enables quick rejection of most of the compoundsas irrelevant, and approves only a small fraction for continuedresearch. The present invention is suitable for very thorough andinformative HTS assays, in the sense that it provides real-timemeasurement of translation of specified proteins in viable cells.

Thus, functional activity of a compound on a specific cell type can beusefully studied by subjecting it to a translation monitoring assay ofthe present invention. A cell line with tagged tRNA is cultured andplaced in a multi-well plate. This can have a 96-well plate format, a384-well plate format or any other format compatible with automatedscreening. The wells in the plate need to be optically amenable fordetection.

A robot administers one compound from the library into each well, andsignal detection is performed. A suitable sampling regime should beadopted. As an illustrative example, a measurement can be taken for 30seconds every 10 minutes for a total of one hour. Other regimes canoptionally be also used. The effect of the compound on translationactivity can thus be detected.

It is understood by the skilled artisan that while various options (ofcompounds, properties selected or order of steps) are provided herein,the options are also each provided individually, and can each beindividually segregated from the other options provided herein.Moreover, steps which are obvious and known in the art that willincrease the sensitivity of the assay are intended to be within thescope of this invention. For example, there may be additional washingsteps, blocking steps, etc. It is understood that the exemplaryembodiments provided herein in no way serve to limit the true scope ofthis invention, but rather are presented for illustrative purposes. Allreferences cited herein are expressly incorporated by reference in theirentirety.

The following examples are presented in order to more fully illustratesome embodiments of the invention. They should, in no way be construed,however, as limiting the broad scope of the invention.

Examples Example 1 Labeling Two Parts of the Translational Machinery asa FRET Pair

Two different tRNA molecules are labeled as a FRET pair. For example,some tRNAs (FIG. 1) can be labeled with a donor fluorophore, and otherswith a corresponding acceptor fluorophore such as Cy3 (excitation andemission peaks are 550 and 570 nm, respectively) and Cy5 (excitation andemission peaks are 650 and 670 nm, respectively); when translation isactive, such tRNAs are immobilized in two adjacent sites (A and P or Pand E) of the ribosome, thereby producing a FRET pair which producesmeasurable FRET signals. These signals indicate that the A and P sitesare populated with labeled tRNAs. A small signal indicates that a lowpercentage of the A and P sites are populated, and therefore that thetranslation apparatus is in a state of low production rates. Additionalexemplary, non-limiting FRET combinations are listed in Table 3.

TABLE 3 Exemplary FRET combinations. Donor fluorophore Acceptorfluorophore Cy3 Cy5 Rhoadmine 110 Cy3 Cy3 Cy5.5

Three docked tRNAs are shown in FIG. 1. The first 32 is in the A(Aminoacyl) site; the second 33 in the P (Peptidyl) site, and the aminoacid it carries is at this point connected to the nascent peptide; thethird 34 is in the E (exit) site, it has been discharged from the aminoacid and will be ejected shortly from the ribosome. The heavy line 30indicates the mRNA being translated, and the dotted line 45 representsthe polypeptide being synthesized, tied into the Peptidyl position.

The main stages of elongation are as follows. Stage 1: Codonrecognition. A tRNA molecule carrying an amino acid binds to a vacantA-site, while the nascent polypeptide is attached to the P-site. Stage2: Peptide bond creation. A new peptide bond is created and thepolypeptide chain is moved to the A-site. Stage 3: Translocation. Theribosome translocates a distance of 3 nucleotides with respect to themRNA, the two tRNA units and the polypeptide chain. Stage 4: the cyclerepeats itself until a stop codon is reached.

Three types of tRNA are shown with respect to fluorescent labeling. ThetRNAs 40 and 43 are unlabeled. tRNAs 33 and 42 (marked with verticallines) are labeled as FRET donors. tRNAs 41 and 32 (marked withhorizontal lines) are labeled as FRET acceptors. When freely diffusing(as in the case of 41 and b), the chance of a FRET pair forming for ameasurable length of time is negligible. However, when a pair isimmobilized on the ribosome (as in the case of 32 and 33), a FRET pairis formed for about 500 milliseconds (in eukaryotes), which issufficient for detection.

The larger the number of active ribosomes, the larger the probability ofjuxtaposition of such pairs, and the larger the FRET signal. Inaddition, signal variability can be used to estimate the concentrationof active ribosomes. Also, with a microscope, subcellular localizationof protein synthesis can be quantitatively estimated. tRNA pairs thatare not immobilized in such a way either diffuse in he cytoplasm or elseare bound to non-labeled molecules such as translation factors oramino-acyl synthetases, and therefore do not create FRET pairs, yieldingno measurable signal. This basic principle holds for any choice of FRETpairs.

Example 2 Introduction of the Labeled tRNAs into CHO Cells

Labeled tRNAs are transfected into CHO cells using TransMessengertransfection reagent (Qiagen, Hilden, Germany) according to themanufacturer's protocol. Transfected cells are placed under a microscopeequipped for single molecule detection (Zeiss, Oberkochen, Germany) withan image acquisition device operable at a sufficient rate (10-100 framesper second), and computational units that can acquire and analyze theresulting images and data (FIG. 2). Translation of the tRNA of interestis measured.

For high-throughput screening, transfected cells are cultured in a96-well plate format, compatible with automated screening. A robotadministers one compound out of the library being screened into eachwell and translation detection is performed. A suitable sampling regimeis adopted. The effect of the compound on translation activity isdetected in comparison with negative control signal.

Example 3 Diagnostic Applications

A selected yeast tRNA is labeled with donor fluorophore and stored.Another selected yeast tRNA is labeled with acceptor fluorophore andstored. Prior to the assay, two aliquots of donor- and acceptor-labeledtRNA are mixed. The mixture is transfected into the cells to bediagnosed, for example, by using the transfection kit RNAiFect™ (Qiagen,Hilden, Germany). The cells may be human cells, for example, human cellsobtained from a tissue removed by biopsy. The transfected cells areintroduced into a 96 well-plate. Signals are collected from the platesusing a fluorescent plate reader and are subjected to computerizedanalysis/es. Typically, the parameters derived for the analysis are:average signal strength, average signal deviation, or percentage oflabeled tRNA of a given species in each well. These parameters aremonitored over time and in response to treatment. Values before and/orafter treatment are compared to known standards to infer clinicalparameters of the cells.

Example 4 Formation and Translocation of a Pre-Translocation (PRE)Complex

Three samples of PRE complex, containing tRNA^(met) in the ribosomalP-site and fMetPhe-tRNA^(Phe) in the ribosomal A-site made withmRNA-fMetPheLys programmed ribosomes were formed in parallel, byaddition of: i) both fMet-tRNA^(fmet)(Cy5) and Phe-tRNA^(Phe)(Cy3) (thedonor-acceptor or DA sample); ii) unlabeled fMet-tRNA^(fmet) andPhe-tRNA^(Phe)(Cy3) (the donor alone or DU sample); and iii)fMet-tRNA^(fmet)(Cy5) and unlabeled Phe-tRNA^(Phe) (the acceptor aloneor UA sample). These complexes, which result in fMetPhe formation, werepurified by ultracentrifugation through a sucrose cushion prior to theirutilization in the fluorescence measurements described below. Thestoichiometries of fMetPhe formed, and of [³H]-Phe and [³⁵5]-fMetcosedimenting with the ribosome in the PRE complexes, are very similarwhether using Cy3-labeled or unlabeled Phe-tRNA^(Phe) (DU and DA samplesvs. UA sample) or Cy5-labeled or unlabeled fMet-tRNA^(fmet) (UA and DAsamples vs. DU sample). These results provide a convincing demonstrationof the functionality of the Cy-labeled tRNAs in binding to the ribosomeand participating in formation of a dipeptide, as part of PRE complexformation.

Fluorescence spectra of the three PRE samples (FIG. 5A) provide clearevidence of FRET in the DA sample, as shown by the increase in acceptorand decrease in donor fluorescent intensities relative to the A and Dsamples, respectively. Addition of EF-G-GTP to each of the samplesleading to post-translocation (POST) complex formation results in amarked decrease in FRET efficiency, as evidenced by the decreases in thedifferences observed comparing the DA sample with both the UA and DUsamples (FIG. 5B). This decrease in FRET efficiency is consistent withan increase in the distance between the dihydrouracil positions oftRNA^(Phe) and tRNA^(fmet) as these two tRNAs move from occupying the A-and P-positions, respectively, in the PRE complex, to occupying the P-and E-positions, respectively, in the POST complex.

Example 5 In-Vitro Protein Sequencing

In this experiment the dynamics of a single ribosome translating a knownmessage in a cell-free system under a single-molecule detection systemwas followed. FIGS. 6A and 6B show events excised from traces of singleribosome experiments. The ribosome was translating the message MRFVRFVRF(FIG. 6A) and MFRVFRVFR (FIG. 6B). tRNA^(Avg) was labeled with Cy3(donor) and tRNA^(Phe) was labeled with Cy5 (acceptor). The traces showthe donor emission under donor excitation (legend: donor), acceptoremission under acceptor excitation (legend: acceptor) and FRET, orsensitized emission, which is acceptor emission under donor excitation(legend: FRET).

In FIG. 6A, an event is shown where a tRNA^(Phe) joins an incumbenttRNA(Arg). Acceptor and FRET traces increase instantaneously andsimultaneously at frame 290 as the tRNA^(Phe) binds; then donor and FRETdisappear, again instantaneously and simultaneously at frame 298 as thetRNA^(Avg) dissociates.

In FIG. 6B, an event is shown where a tRNA^(Avg) joins an incumbenttRNA^(Phe). Donor and FRET traces increase instantaneously andsimultaneously at frame 45 as the tRNA^(Avg) binds; then acceptor andFRET disappear at frame 80 as the tRNA^(Phe) dissociates, simultaneouslywith increase of donor intensity due to dissociation of the FRET pair(leaving the entire energy with the donor).

Example 6 Live Cell tRNA:tRNA FRET Imaging

To demonstrate the feasibility of the assay system of the invention,bulk yeast tRNA labeled with rhodamine 110 as donor and with Cy3 asacceptor were used. Fluorescent labeled tRNA were then introduced intolive HEK-293, HeLa and CHO cells, employing a transfection protocol. Oneday prior to the transfection experiment, the cells were seeded onfibronectin-coated glass slides in a 24-well plate. On the followingday, the growth medium was removed and 0.5 ml of fresh medium was addedto each well containing the sub-confluent monolayers. 10 minutes later,0.1 ml of the labeled-tRNA mixed with the transfection reagent andserum-free media were added to each well. The plate was incubated at 37°C. in a 5% CO₂ incubator for 6-8 hr, followed by fixation with 4%paraformaldehyde for 20 min and mounting using fluorescent mountingmedium. The fixed cells on glass slides were then visualized employing aspinning disk confocal microscope. Stable GFP expressing cells showedincreased GFP fluorescence, indicating increased protein synthesis ratesfor 2-36 hours following transfection with tRNA.

In order to determine the imaging parameters to be employed in thefollowing experiments, including the degree of non-specific signalstemming from the cross-stimulation of different fluorophores, cellswere transfected with donor only and with acceptor only, and imaged inthe exact conditions employed for the FRET measurement. The calibrationcoefficients for computing the NFret (corrected FRET) image wereNFret=FRET−0.011*donor−0.004*acceptor. In addition to the basicidentification of a true FRET signal, indicative of the proximity of thelabeled tRNAs when immobilized in the ribosome, cycloheximide wasutilized to freeze the dynamic process of the functionally importantchange in localization of the tRNAs. Imaging of the same cells prior toand following cycloheximide treatment, confirmed the expected increasein FRET signal intensity in both HEK and CHO cells. Furthermore, thesignal obtained in CHO cells was markedly stronger than the one obtainedin HEK cells under similar transfection and imaging conditions. This isan indication of the correlation of signal intensity with synthesisrates, as CHO cells are known to be strong protein producers.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingcurrent knowledge, readily modify and/or adapt for various applicationssuch specific embodiments without undue experimentation and withoutdeparting from the generic concept, and, therefore, such adaptations andmodifications should and are intended to be comprehended within themeaning and range of equivalents of the disclosed embodiments. Althoughthe invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

1-60. (canceled)
 61. A tRNA FRET pair capable of reading at least onepredetermined codon pair in an mRNA sequence encoding a protein ofinterest; and wherein one tRNA member of each tRNA pair is labeled witha donor fluorophore and the other tRNA member is labeled with anacceptor fluorophore.
 62. The tRNA FRET pair according to claim 61,wherein said at least one codon pair occurs in said mRNA sequence at afrequency that is at least five-fold higher than the general frequencyof said at least one codon pair in a reference proteome ortranscriptome.
 63. The tRNA FRET pair according to claim 62, whereinsaid at least one codon pair occurs in said mRNA sequence at a frequencythat is at least ten-fold higher than the general frequency of said atleast one codon pair in a reference proteome or transcriptome.
 64. ThetRNA FRET pair according to claim 61, wherein the enrichment factor ofsaid at least one codon pair is at least 6.0.
 65. The tRNA FRET pairaccording to claim 61, wherein each tRNA of said tRNA pair correspondsto one amino acid such that said each tRNA corresponds to a tRNA fromthe complete set of isoacceptor tRNAs specific for said one amino acid.66. The tRNA FRET pair according to claim 61, wherein said pair isselected from: Cysteine-Leucine, Tryptophan-Tryptophan,Cysteine-Tryptophan, Cysteine-Phenylalanine, Asparagine-Tryptophan,Tyrosine-Tryptophan, Arginine-Alanine, Proline-Arginine,Tyrosine-Serine, Arginine-Arginine, Alanine-Tyrosine, Arginine-Serineand Tyrosine-Phenylalanine.
 67. The tRNA FRET pair according to claim66, wherein the pair Cysteine-Leucine is for measuring the translationof filgrastim; the pair Cysteine-Phenylalanine is for measuring thetranslation of somatotropin; the pair is selected from Arginine-Alanine,Proline-Arginine and Tyrosine-Serine for measuring the translation ofALT1; the pair is selected from Arginine-Arginine, Alanine-Tyrosine andArginine-Serine for measuring the translation of ALT2; the pair isAspargine-Tryptophan for measuring the translation of etanercept; andthe pair is Tyrosine-Tryptophan for measuring the translation ofcetuximab.
 68. The tRNA FRET pair according to claim 61, wherein onetRNA member of said tRNA FRET pair is capable of reading synonymouscodons encoding a particular amino acid.
 69. The tRNA FRET pairaccording to claim 61, wherein said tRNA pair corresponds to a specificpair of adjacent amino acids occurring at said protein of interest.